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Malignant mixed Mullerian tumors of the uterus: histopathological evaluation of cell cycle and apoptotic regulatory proteins
Rani Kanthan1,
Jenna-Lynn B Senger1 &
Dana Diudea1
World Journal of Surgical Oncology volume 8, Article number: 60 (2010) Cite this article
The aim of our study was to evaluate survival outcomes in malignant mixed Mullerian tumors (MMMT) of the uterus with respect to the role of cell cycle and apoptotic regulatory proteins in the carcinomatous and sarcomatous components.
23 cases of uterine MMMT identified from the Saskatchewan Cancer Agency (1970-1999) were evaluated. Immunohistochemical expression of Bad, Mcl-1, bcl-x, bak, mdm2, bax, p16, p21, p53, p27, EMA, Bcl-2, Ki67 and PCNA was correlated with clinico-pathological data including survival outcomes.
Histopathological examination confirmed malignant epithelial component with homologous (12 cases) and heterologous (11 cases) sarcomatous elements. P53 was strongly expressed (70-95%) in 15 cases and negative in 5 cases. The average survival in the p53+ve cases was 3.56 years as opposed to 8.94 years in p53-ve cases. Overexpression of p16 and Mcl-1 were observed in patients with longer survival outcomes (> 2 years). P16 and p21 were overexpressed in the carcinomatous and sarcomatous elements respectively. Cyclin-D1 was focally expressed only in the carcinomatous elements.
Our study supports that a) cell cycle and apoptotic regulatory protein dysregulation is an important pathway for tumorigenesis and b) p53 is an important immunoprognostic marker in MMMT of the uterus.
Malignant mixed Mullerian tumors (MMMT) of the uterus are rare, high-grade neoplasms comprising only 1-2% of uterine cancers [1] and 3-5% of all uterine malignancies [2]. They are the most common variety of mixed epithelial and non-epithelial endometrial tumors, with a clinically aggressive course [3, 4]. Stage of the disease and the depth of myometrial invasion are recognized as important prognostic factors [5–7]. Two-year survival rates have been reported at 53% in stage I (confined to uterine corpus) and 8.5% in stages II (cervical metastases) and III (pelvic metastases), with none reported in Stage IV [8]. Common in the uterus, this tumor may arise in the ovaries, fallopian tubes and vagina [5, 9]. Histologically, MMMT is a biphasic tumor composed of both epithelial (carcinoma) elements and mesenchymal (sarcoma) elements; though, which component is responsible for the tumor's aggressive biological behavior remains undetermined [2, 10–15].
Three theories proposed to ascertain this tumor's histiogenesis include that MMMTs may be 1) collision tumors, 2) combination tumors, or 3) composition tumors. Immunophenotypical and ultrastructural studies that favor the third theory explain MMMTs as being monoclonal in origin, with diverse carcinomatous and sarcomatous elements that can be homologous (histologically native, worse prognosis) or heterologous (foreign, better prognosis) to the organ [13, 15–18]. MMMTs occur in postmenopausal women and usually present in an advanced stage with abdominal pain, distension, and atypical spotting/bleeding [18–21]. While it is presumed that MMMTs arise from pre-existing carcinomas, little is known about the etiopathogenesis of MMMTs. Exposure to radiation, excessive estrogen exposure, obesity, and nulliparity [22, 23] are believed to be associated with MMMT development.
It is usually understood that carcinogenesis is a multistep process that involves defects of the genetic pathways including cell proliferation, cell adhesion, cell death and apoptosis [2]. Cell survival and apoptotic regulatory proteins such as the Bcl-2 family of genes, PCNA, p16, p21, p27, and cyclin D1 are of vital importance to malignant neoplasms in prolonging cell survival. Despite the understanding that cell cycle regulatory protein dysregulation may be involved in numerous malignant tumors [2], there is limited data that explores the role of these oncoproteins with survival data in MMMTs. The aim of this study is to evaluate the role of cell cycle and apoptotic regulatory proteins in the carcinomatous and sarcomatous components of uterine MMMT in relation to clinico-pathological data including survival outcomes.
Twenty-three cases of uterine MMMT were identified from the records of the Saskatchewan Cancer Agency (1970-99). The original slides and paraffin blocks were retrieved and reviewed to confirm the diagnosis as seen in Figures 1A and 1B. A representative block was chosen for detailed histological and immunohistochemical study with the antibodies as listed in Table 1. EMA, Bcl-2, Ki67, PCNA, Bad, Mcl-1; bcl-x, bak, mdm2, bax, p16, p21, p27, p53 and Cyclin D1 expression were evaluated by the standard avidin-biotin complex method with positive and negative controls as per standard laboratory protocol. Immunostaining results were scored on a semi-quantitative scale including staining intensity and percentage of positive cells. The extent of immunostaining was divided into four categories according to the percentage of immunostained neoplastic cells: < 25% (1+), 25-50% (2+), 50-75% (3+), and > 75% (4+). In addition, the qualitative intensity of immunostaining of the tumor cells was quantitatively scored into three categories: weak (1+), moderate (2+), and strong (3+) as seen in Figure 1C. The intensity of the endothelial cell staining served as an internal control. A combined immunoreactivity score was calculated by multiplying the score for the percentage x the score of intensity, resulting in a combined score that ranged from 0 to 12. Scores 4 and above were considered positive for the purposes of this study.
Histopathological Evaluation with Immunohistochemical Staining. A: Hematoxylin-eosin stain (original magnification ×250). The star (*) indicates the malignant heterologous component of uterine MMMT. B: Hematoxylin-eosin stain (original magnification ×250). The arrows indicate the malignant epithelial component of uterine MMMT. C: Staining with Bax antibody (original magnification ×250). The expression of Bax antibody is diffuse with the thin arrowhead indicating weak staining, the thick arrowhead indicating medium staining, and the tailed arrow indicating strong staining. D: Staining with p53 antibody(original magnification ×250). The star (*) indicates the negatively stained heterologous sarcomatous element, and the arrow indicates positive staining in the epithelial component. E: Staining with p16 antibody (original magnification ×250). The star (*) indicates the negatively stained region, and the arrow indicates the strong positive staining in the malignant epithelial glands. F: Staining with Cyclin D1 antibody (original magnification ×250). The star (*) indicates the negatively stained heterologous sarcomatous element, while the arrow indicates a focus positive staining in the adjacent epithelial component.
Table 1 Clone, dilution, and source of antibodies used for the immunohistochemical analysis in this study
Clinical data such as disease free survival, overall survival, family history of cancer, past medical history, and treatment protocols were obtained by detailed analysis of their case records. Statistical analysis was preformed with Kruskal-Wallis, Fischer's Exact Test, and a Mann-Whitney post hoc test for independent data. A p value of ≤ 0.05 was regarded as statistically significant.
This study was conducted with ethics approval from the University of Saskatchewan Biomedical Research Ethics Review Committee.
Demographics and Clinical Measures
Table 2 and Figure 2 list the various demographic and clinico-pathological data of 23 patients with uterine MMMT. The majority of patients (39.1%) were 71-80 years old, followed by 61-70 years (26.1%). 18 patients (78.3%) presented with postmenopausal bleeding. Histologically, 11 patients (47.8%) had homologous elements, while 10 (43.5%) had heterologous elements. 10 patients (43.5%) were Stage I, two (8.7%) Stage II, three (13.0%) Stage III and seven (30.4%) Stage IV. Myometrial depth of invasion was superficial in 43.5% of patients, and deep in 30.4%. Metastases were present in 43.5% of patients at presentation in the liver, ovaries, fallopian tube, abdomen, peritoneum, ommentum, bladder, and iliac lymph nodes. Five cases (21.7%) exhibited pelvic metastasis. Lung and cervical metastasis were present in 2 patients (8.7%). Management protocols included surgery (20 patients, 87.0%), chemotherapy, (2 patients, 8.7%) and radiation therapy (14 patients, 60.9%).
Table 2 Patient demographics with clinico-pathological data
Demographic and Clinical Data in Relation to 2 Year Survival Data. X-axis displays: age, postmenopausal bleeding, homologous elements, stage III/IV, depth of invasion, and metastasis. Y-axis displays: survival outcome data-including overall survival (purple bars), two-year survival (maroon bars), and less than two-year survival (yellow bars). * p < 0.05 based on Fisher's Exact Test.
Relationship with Survival Time
One of the main goals of this study was to determine the prognostic value of the demographic and clinico-pathological data to the immunohistochemical expression of the biological markers studied. The stage of the disease (Table 2 and Figure 2) indicates the proportion of subjects surviving 2-years. Of the 23 cases, 3 (13.0%) were Stage III, and 7 (30.4%) Stage IV. 77.8% of patients in Stage III/IV survived less than two years while 23.1% had longer survival outcomes. This inference of survival data may not be truly reflective as treatment protocols were not standardized across all cases studied.
Cell cycle and apoptotic regulatory proteins were analyzed for statistical significance as possible prognostic indicators. Two cell cycle proteins, Mcl-1 and p16, were found to be statistically significant. Of the 23 patients, 8 (34.8%) were positive for Mcl-1. As seen in Figure 3, survivors of 2 years, 53.8% had a positive Mcl-1 expression, while only 10.0% of those cases that did not survive showed positive Mcl-1 expression. Similar results were obtained for p16. Seven cases (30.4%) were positive for this cell cycle protein. Of the patients who survived two years, 46.2% exhibited positive p16 expression, while only 10.0% of those who did not survive 2 years did. P53 was strongly expressed (70-95%) in 15 cases and negative in 5 cases. The average survival in the P53+ve cases was 3.56 years as opposed to 8.94 years in P53-ve cases.
Cell Cycle and Apoptotic Regulatory Proteins in Relation to 2 Year Survival Data. Statistical significance: *p < 0.05, †p < 0.10.
Protein Markers
The histological samples from these 23 cases were analyzed for the presence of various biological markers, including both cell cycle and apoptotic regulatory proteins. The cell cycle proteins studied include p16, p21, p27, p53, Cyclin D1 and Ki67. As seen in Table 3, p16 was positively expressed in 7 cases (30.4%), p53 in 15 cases (65.2%), and Ki67 in 10 patients (43.5%). There was no overexpression of p27. Cyclin D1, though predominantly negative, was expressed focally in the carcinomatous elements.
Table 3 Immunohistochemical expression of cell cycle and apoptotic regulatory protein antibodies
The apoptotic regulatory proteins, which were analyzed, include the following: Bad, Bak, Mcl-1, Bcl-2, Bcl-x, Mdm-2, and Bax. Bad was overexpressed in 82.6% of cases (19 patients) and Bak was positive in 73.9% (17 patients). Positive Bax expression was seen in 60.9% (14 patients). The remaining proteins had less than 50% positive expression (Figure 3).
Uterine MMMT are malignant neoplasms that contain atypical malignant endometrial glands admixed with heterologous or homologous sarcomatous elements [2, 10–14] with the dominant element often being the epithelial component yet distinct from endometrial carcinoma[13]. Occurring predominantly in postmenopausal women [24, 25], the prognosis of MMMT is generally worse than endometrial carcinoma. These are rare tumors with an annual incidence of 2/100 000 women, comprising 2-5% of all gynecologic tumors [26, 27]. Five-year survival rates are reported between 18-39%. Many cases (70%) present with advanced disease (Stage III/IV), contributing to poor survival rates [21]. This tumor spreads locally within the pelvic cavity and distally to the regional lymph nodes, lungs and liver. DiSaia et al. [28] reported a 2 year survival rate of 53% in patients with tumors confined to the uterine corpus (Stage I), which dropped to 8.5% if the disease had extended into the cervix, vagina or parametrium (Stages II/IIII). Less than two year survival was seen in Stage IV disease [4], similar to other studies, with 5-year disease-free survival rates being: Stage 1 56%, Stage II 31%, Stage III 13%, Stage IV 0% [29]. Our study revealed similar trends. Three cases diagnosed as Stage III did not survive beyond two years. 44.4% of seven cases diagnosed as Stage IV did not survive beyond two years. However, three stage IV patients had longer survival outcomes in contrast to published literature [5]. This finding may be related to small sample size.
The stage of the disease and the depth of myometrial invasion were statistically significant prognostic factors in our study, similar to reports by other authors [5, 7]. Other demographic and clinico-pathological data including age, postmenopausal bleeding, histological type, metastasis, and treatment modalities were not found to be statistically significant in our study.
Uterine MMMT metastasizes similar to endometrial carcinoma of the uterus, with recurrence occurring commonly in the upper abdomen with occasional distant spread [28]. In our study, 43.5% developed some form of metastasis. Metastases occurred in 70.0% of subjects who did not survive 2 years while longer survival time was associated with lowered metastases (23.1%). This is statistically significant, and indicates that the presence of metastasis at presentation is a strong prognostic indicator for overall survival outcomes. The exact nature of whether the carcinomatous or the sarcomatous element is the more aggressive component and therefore has greater propensity for metastases remains an unresolved and controversial issue [13].
Uterine MMMTs consist of carcinomatous (CA) and sarcomatous components (SA). Histopathological evaluation of which component is responsible for biological tumor aggressiveness has not been greatly explored. Yoshida et al [8] reported a higher microvessel density in the carcinomatous region and a higher apoptotic index in the sarcomatous areas, from which they concluded that the carcinomatous components likely played an important role in the aggressive biological behavior of MMMT [22]. This biological behavior is similar to endometrial carcinoma with which they share common etiopathological factors.
Cell proliferation, including initiation, promotion and progression (invasion and metastasis), plays a central role in the multistep process of carcinogenesis. Replication of damaged DNA is necessary to fix base substitutions, frame shift mutations, allelic deletion and induction of chromosomal translocations. Cancer cells commonly demonstrate errors in these pathways during cell cycle proliferation. Proliferative markers such as Ki67 provide an index of cells in the Go/G1 pool of cycling cells [30]. Higher fractions of these cells represent an increased number of cells subjected to genetic instability. In our study Ki67 was overexpressed (50-80% positive cells) in 10 cases with no statistical difference between the carcinomatous and the sarcomatous areas. This lack of difference in antigen expression between the epithelial and the sarcomatous areas is consistent with other studies [2]; thus supporting that the histogenesis of this tumor is probably from a single pluripotential malignant clone with divergent histologic differentiation [2].
Mutations in the p53 gene (tumor suppresser and gatekeeper) remain one of the commonest genetic lesions found in human cancers. This occurs in both the carcinomatous and sarcomatous elements of uterine MMMT [31–35]. Such mutations result in abnormal protein expression, with increased intracellular accumulation due to an increased half life which is easily detected by immunohistochemical methods [36–38]. In our series, overexpression of p53 (70-95%) was negative in 8 cases and positive in 15 cases, predominantly in the sarcomatous regions as seen in Figure 1D. The average survival time in p53 positive cases was 3.56 years as opposed to 8.94 years in the negatively stained cases. The cohort of the positive cases was also predominantly older (71-90 years). Though p53 positive expression cases in our study were not statistically significant in regard to survival beyond two years, it was interesting to note that p53 negative cases were associated with an improved clinical outcome. Overexpression of p53 has been linked to decreased survival in several other malignancies. These include human soft tissue sarcomas [39] and some cases of breast, lung and colorectal carcinomas [40]. Such trends clearly suggest that p53 may play a key role in the multistep evolution of disease progression in MMMT [41, 42]; however, it is postulated like in pulmonary carcinosarcoma to be a late event in the disease progression with resultant better survival outcomes in those cases that have not yet acquired the defect [43]. In this context, further studies of p53 mutation analysis by PCR-SSCP with sequencing will help confirm these observed trends.
Central to the cell cycle regulatory protein machinery is a family of serine-threonine kinases, the cyclin dependent kinases (CDKs). These kinases are activated by cyclins D and E and inactivated by CDK inhibitors (CDKIs) including: p27, p16, and p21[44].
p16 specifically inhibits the cyclin D1-CDK4/6 complex and, along with the main substrate, forms the retinoblastoma gene product (pRb), which is the most important regulatory pathway involved in the G1/S transition [45, 46]. Frequent expression of p16 in primary tumors suggests that the p16 protein is involved in the development of these lesions [2]. Uncontrolled tumor cell proliferation is frequent in tumor cells with the progression of a normal cell to a transformed tumor cell involving many genetic events that include the checkpoints of the cell cycle machinery [44, 47]. Overexpression of p16 is believed to be the result of mutated p16 gene product and/or an accumulation due to decreased turnover of the protein [2]. Overexpression of p16 in the carcinomatous regions of MMMT (Figure 1E) with inverse expression of p21 in these regions denotes an upregulation of p16. The latter is also supported by a failure to express cyclin D1 in the majority of the tumor cells with focal expression only in the carcinomatous elements (Figure 1F). This supports the theory of a damaged regulatory pathway wherein p16 predominantly inhibits cyclin-D1 associated kinase activities [48]. P16 could also mediate contact inhibition of growth and thus may be responsible for the invasive powers of the neoplasm. It is interesting to note that many of the initial metastases in MMMT consist entirely of carcinomatous elements, thus supporting the theory that the carcinomatous component is perhaps responsible for the initial biological aggressiveness of the tumor. This change over time is also reported in the literature as loss of p16 in some cases of MMMTs when they recurred [2]. In our study cases 46.2% of subjects who survived 2 years had positive p16 overexpression in contrast to a lower expression in 10% of cases with less than two year survival.
Cell death plays an important role in normal tissue homeostasis wherein the finite balance between new cell productions caused by cell division is offset by cell loss in tissues capable of cell renewal. Cells that succumb to this mechanism of cell death undergo characteristic morphological and biochemical changes that are termed apoptosis. Apoptosis is one aspect of mammalian cell behavior, which is of central importance in growth and development and plays a key role in tumor-oncogenesis. The three key features of apoptosis and cell survival are related to inciting the signal transduction pathways of the bcl-2 family of genes and the ICE family of proteases. These components interact with other cell cycle related genes such as p53. The central role of the Bcl-2 family of genes in the regulation of apoptosis has been convincingly demonstrated [49–56]. The interaction of Bcl-2 family of proteins is viewed in terms of two mechanisms: a) at least two rheostats - the Bcl-2/Bax ratio and the Bcl-xL/Bcl-xs ratio and b) a quaternary complex involving anti-apoptotic protein, pro-apoptotic protein, caspase and Apf-1 equivalent protein. The susceptibility to apoptosis is likely to be determined by the ratio of the positive regulators (Bak, Bax, Bcl-xs) to negative regulators (Bcl-2, Bcl-xL, Mcl-1 and A1) [57]. The role and contribution of each of these factors is likely to be specific for different cells and tissues. The function of Bcl-2 protein is dependent on post-translational modification, specifically phosphorylation of serine/threonine residues.
Therefore, mere overexpression of the protein does not provide complete information. Also, the finding that Bcl-2 is not expressed in a variety of tumors indicates that other apoptosis-modulating factors, especially Bcl-xL/Bcl-xs, may play a role [58–63]. In view of the dimeric interactions of Bcl-2 family proteins and interaction with other apoptosis regulators, assessment of one protein alone is unlikely to provide an understanding of apoptosis regulation. Deregulation of the biochemical pathways that control physiological cell death can contribute to neoplastic cell expansion by preventing or delaying normal cell death. One of the critical regulators of apoptosis is the protein encoded by the Bcl-2 gene [64, 65]. Although the exact biochemical mechanism of Bcl-2 remains enigmatic, the Bcl-2 protein appears to control a distal step in the final common pathway for apoptotic cell death. Recently, a family of genes have been identified whose encoded proteins share amino acid sequence homology with Bcl-2. Some of these genes function as blockers of cell death while others promote apoptosis [56, 57, 66]. Among this multigene family, the protein encoded by the Bax gene has emerged as a central regulator [65, 67, 68]. The Bax protein is a promoter of cell death, while others such as Bcl-x and Mcl-1 function as suppressors of cell death. Further, it has been proposed that the relative sensitivity of cells to apoptotic stimuli is governed by the ratio of Bax: Bcl-2 and other antiapoptotic Bcl-2 family proteins [58]. Gene transfer experiments indicate that Bax is a regulator, not an effector of the programmed cell death pathway. As a result, it should be possible to induce apoptosis even in the absence of Bax provided that the apoptosis stimulus is of sufficient strength. Since Bcl-2 can abrogate apoptosis promoted by Bax, it is possible that it is Bax that mainly regulates the threshold of loss of apoptosis. P53 is known to regulate Bax expression, with inactivation of p53 leading to reduced Bax protein levels [69, 70]. Bax mutations and resistance to apoptosis have been described in stomach, pancreas, endometrium, hemopoietic malignancies, and a subset of colon and lung cancers [71, 72] indicating that inactivating Bax mutations may play an important role in tumor progression in these cancers.
In our study, all cases demonstrated diffuse expression of Bax, Bad and Bak proteins in contrast to weak or negative expression of Mcl-1, MDM2 and Bcl-x. This supports the existence of apoptosis protein dysregulation in these lesions. The exact biochemical mechanism of such dysregulatory pathways remains unclear. Expression of Mcl-1 was not statistically significant in regard to the two year survival data; though, Mcl-1 was expressed in a higher proportion of cases that survived 2 years. This finding needs further investigation in larger samples.
Currently, there is no consensus treatment guidelines related to improved survival outcomes. The rarity of this tumor limits the potential for large clinical trials [73]. Nevertheless, the persistent high mortality rate and high recurrence rate [73] with no significant improvement in patient survival during the past 40 years [24] demands the attention and time of researchers in a fight to improve treatment modalities and widen the understanding of uterine MMMT.
Our study supports that cell cycle and apoptotic regulatory protein dysregulation is an important pathway for tumorigenesis. Apoptotic protein dysregulation may result in epigenetic silencing of cell cycle pathways resulting in disarrayed/differential growth patterns. Future genetic analysis of Bad/bax/bak pathway may provide further insight in elucidating this mechanism.
In uterine MMMT p53+ve tumors occur in older women with a short mean survival while p53-ve tumors occur in younger women with longer survival. Such trends suggest that p53 may be an important immunoprognostic marker in this neoplasm that warrants further exploration.
Both p16 and Mcl-1 expression were associated with longer survival (>2 years). Further research regarding these cell cycle regulatory proteins will shed light into their possibility as future predictive/prognostic markers.
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Gagner JP, Mittal K: Malignant Mixed Mullerian Tumor of the Fimbriated End of the Fallopian Tube: Origin as an Intraepithelial Carcinoma. Gynecologic Oncology. 2005, 97 (1): 219-222. 10.1016/j.ygyno.2004.11.053.
Geisler JP, Geisler HE, Wiemann MC, Zhou Z, Miller GA, Crabtree W: p53 Expression as a Prognostic Indicator of 5-Year Survival in Endometrial Cancer. Gynecol Oncol. 1999, 74 (3): 468-471. 10.1006/gyno.1999.5482.
Liu FS, Kohler MF, Marks JR, Bast RC, Boyd J, Berchuck A: Mutation and Overexpression of the p53 Tumor Suppressor Gene Frequently Occurs in Uterine and Ovarian Sarcomas. Obs and Gynecol. 1994, 83 (1): 118-123.
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Huang LW, Chou YY, Chao SL, Chen TJ, Lee TT: p53 and p21 Expression in Precancerous Lesions and Carcinomas of the Uterine Cervix: Overexpression of p53 Predicts Poor Disease Outcome. Gynecol Oncol. 2001, 83 (2): 348-354. 10.1006/gyno.2001.6397.
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Ramirez PT, Gershenson DM, Tortolero-Luna G, Ramondetta LM, Fightmaster D, Wharton JT, Wolf JK: Expression of Cell-Cycle Mediators in Ovarian Cancer Cells after Transfection with p16, p21, and p53. Gynecol Oncol. 2001, 83 (3): 543-548. 10.1006/gyno.2001.6438.
Munirajan AK, Kannan K, Bhuvarahamurthy V, Ishida I, Fujinaga K, Tsuchida N, Shanmugam G: The Status of Human Papillomavirus and Tumor Suppressor Genes p53 and p16 in Carcinomas of Uterine Cervix from India. Gynecol Oncol. 1998, 69 (3): 205-209. 10.1006/gyno.1998.4991.
Shapiro GI, Edwards CD, Ewen ME, Rollins BJ: p16INK4A participates in a G1 arrest checkpoint in response to DNA damage. Mol Cell Biol. 1998, 18 (1): 378-387.
Dimitrakakis C, Kymionis G, Diakomanolis E, Papaspyrou I, Rodolakis A, Arzimanoglou I, Leandros E, Michalas S: The Possible Role of p53 and bcl-2 Expression in Cervical Carcinomas and Their Premalignant Lesions. Gynecol Oncol. 2000, 77 (1): 129-136. 10.1006/gyno.1999.5715.
Thompson C: Apoptosis in the Pathogenesis and Treatment of Disease. Science. 1995, 267 (5203): 1457-1461. 10.1126/science.7878464.
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Kerr JF, Winterford CM, Harmon BV: Apoptosis - Its Significance in Cancer and Cancer Therapy. Cancer. 1994, 73 (8): 2013-2023. 10.1002/1097-0142(19940415)73:8<2013::AID-CNCR2820730802>3.0.CO;2-J.
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Crescenzi E, Palumbo G: Bcl-2 Exerts a pRb-Mediated Cell Cycle Inhibitory Function in HEC1B Endometrial Carcinoma Cells. Gynecol Oncol. 2001, 81 (2): 184-192. 10.1006/gyno.2001.6128.
Nakamura T, Nomura S, Sakai T, Nariya S: Expression of bcl-2 Oncoprotein in Gastrointestinal and Uterine Carcinomas and their Premalignant Lesions. Hum Pathol. 1997, 28 (3): 309-15. 10.1016/S0046-8177(97)90129-5.
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Krajewska M, Moss SF, Krajewski S, Song K, Holt PR, Reed JC: Elevated Expression of Bcl-X and Reduced Bak in Primary Colorectal Adenocarcinomas. Cancer Res. 1996, 56 (10): 2422-7.
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Crescenzi E, Criniti V, Pianese M, Tecce MF, Palumbo G: Differential Expression of Antiapoptotic Genes in Human Endometrial Carcinoma: bcl-XL Succeeds bcl-2 Function in Neoplastic Cells. Gynecol Oncol. 2000, 77 (3): 419-428. 10.1006/gyno.2000.5803.
Oltvai ZN, Milliman CL, Korsmeyer SJ: Bcl-2 Heterodimerizes in vivo with a Conserved Homologue Bax, that Accelerates Programmed Cell Death. Cell. 1993, 74: 609-619. 10.1016/0092-8674(93)90509-O.
Gross A, McDonnell JM, Korsmeyer SJ: Bcl-2 Family Members and the Mitochondria in Apoptosis. Genes Dev. 1999, 13 (15): 1899-1911. 10.1101/gad.13.15.1899.
Kokawa K, Shikone T, Otani T, Nishiyama R, Ishii Y, Yagi S, Yamoto M: Apoptosis and the Expression of Bcl-2 and Bax in Patients with Endometrioid Clear Cell and Serous Carcinomas of the Uterine Endometrium. Gynecol Oncol. 2001, 81 (2): 178-183. 10.1006/gyno.2001.6138.
Ouyang H, Furukawa T, Abe T, Kato Y, Horii A: The Bax Gene the Promoter of Apoptosis is Mutated in Genetically Unstable Cancers of the Colorectum Stomach, and Endometrium. Clin Cancer Res. 1998, 4 (4): 1071-4.
Oda K, Arakawa H, Tanaka T, Matsuda K, Tanikawa C, Mori T, Nishimori H, Tamai K, Tokino T, Nakamura Y, Taya Y: p53AIP1, a Potential Mediator of p53-Dependent Apoptosis and Its Regulation by Ser-46-Phosphorylated p53. Cell. 2000, 102 (6): 849-862. 10.1016/S0092-8674(00)00073-8.
Miyashita T, Reed JC: Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 1995, 80 (2): 293-299. 10.1016/0092-8674(95)90412-3.
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Hanaoka T, Nakayama J, Haniuda M, Sato TA: Immunohistochemical Demonstration of Apoptosis-Regulated Proteins Bcl-2 and Bax in Resected Non-Small-Cell Lung Cancers. Int J Clin Oncol. 2002, 7 (3): 152-8. 10.1007/s101470200022.
Wong L, See HT, Khoo-Tan HS, Low JS, Ng WT, Low JJ: Combined Adjuvant Cisplating and Ifosfamide Chemotherapy and Radiotherapy for Malignant Mixed Mullerian Tumors of the Uterus. International Journal of Gynaecological Cancer. 2006, 16 (3): 1364-9. 10.1111/j.1525-1438.2006.00560.x.
This study was partly funded by The Scientific Teaching and Research Grant of the College of Medicine, University of Saskatchewan.
Department of Pathology and Laboratory Medicine, University of Saskatchewan, Saskatoon, SK, Canada
Rani Kanthan, Jenna-Lynn B Senger & Dana Diudea
Rani Kanthan
Jenna-Lynn B Senger
Dana Diudea
Correspondence to Rani Kanthan.
RK is the corresponding, and first author of this manuscript. JLBS contributed to the acquisition, analysis, and interpretation of data. DD provided overall expertise. All authors have read and approved the final manuscript.
Kanthan, R., Senger, JL.B. & Diudea, D. Malignant mixed Mullerian tumors of the uterus: histopathological evaluation of cell cycle and apoptotic regulatory proteins. World J Surg Onc 8, 60 (2010). https://doi.org/10.1186/1477-7819-8-60
Myometrial Invasion
Cell Cycle Regulatory Protein
Sarcomatous Component
Malignant Mixed Mullerian Tumor


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LONDON, U.K. – Fort St. John native Jamie Herrington has been stripped of her title at the 2015 IMMAF World Championships of Amateur Mixed Martial Arts.
An independent tribunal appointed by the International Mixed Martial Arts Federation found that Herrington committed an Anti-Doping Rule Violation at the event in Las Vegas. As result, she was stripped of her medal, and has been banned from competition for four years.
Herrington participated in a number of matches in the tournament back in July, 2015, subsequently winning the gold medal in the 145 lb. weight class. She submitted to a post-bout substances test immediately after one of her matches. Two samples were collected, and then submitted to the World Anti-Doping Agency Laboratory at UCLA for analysis.
The "A" sample showed a positive result for amphetamine, which is a prohibited substance under Section 6 of the WADA Code, and therefore a breach of the IMMAF's Anti-Doping Rules. Herrington was notified of the result on September 3rd, 2015 and received a provisional suspension on that date.
At Herrington's request, her "B" sample was analysed, and also presented a positive result for amphetamine. In reaching a decision on consequences to be imposed, the independent tribunal considered the Athlete's explanation as to how the substance came to be present in her system, whether there was evidence of intention and the Athlete's degree of fault in relation to this violation.
However, as no reductions were found to be applicable, the period of ineligibility imposed is 4 years commencing from the date of provisional suspension. Ms Herrington will be eligible to compete again from 3rd September 2019. Additionally, Ms. Herrington's results from the 2015 IMMAF World Championships are disqualified with all resulting consequences. In this instance this will result in the forfeiture of Ms Herrington's gold medal, disqualification of her results and subsequent revision of medals in that category. Right of appeal to the Court of Arbitration for Sport has not been taken up by the athlete. No orders for costs were sought or made by IMMAF.
Herrington spoke with Energeticcity.ca and continued to proclaim her innocence, stating that she took Nyquil the night before her title fight, as she was not feeling well. She says that the pseudoephedrine that is present in Nyquil and acts as a decongestant is also a stimulant. According to her, the pseudoephedrine caused the false positive result for amphetamine in both of her results. "The people who know me and the friends and fmily that are close to me know that I am not involved in drugs or alcohol. That's what I have to go on at the end of the day," said Herrington. She added that she feels that the four-year suspension was also exceptionally harsh, pointing out to the suspensions handed out to several high-profile UFC fighters for using banned substances were all less than hers.
"We were shocked and disappointed to hear from IMMAF about Jamie's failed drug test. We have always had a no drugs/steroids/gang affiliation rule amongst our fight team in an effort to keep only the highest standard of athlete representing our team.
When receiving the news last year from IMMAF and Team Canada representatives we felt we had no choice but to remove Jamie from our fight team, the gym and any competition hosted by Fivestar. It was a heartbreaking situation for our coaching staff here who offered her a lot of support, training and funding to get her to the national tryouts and worlds just to be disappointed by her decisions.
Although it seems somewhat steep perhaps, We appreciate IMMAF's stance on the punishment as they have been working very hard to have MMA added as an Olympic sport and such standards are necessary.
A full copy of the decision is available here: http://www.immaf.org/wp-content/uploads/2015/09/09.02.16-HERRINGTON.pdf.


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Why 'Max Payne' is one of the best trilogies in gaming.
Mike E, 1 year ago 0
Max Payne. Dearest of all my friends. One pessimistic individual. But I love him, and if you haven't had the experience of playing this trilogy which just turned 20 years old, let me tell you (with no game breaking spoilers) why you're missing out on not just a fantastic game series but a fantastic piece of art.
"I don't know about angels, but it's fear that gives men wings."
– Max Payne, 2001
The first two games of this trilogy, 'Max Payne' and 'Max Payne 2: The Fall of Max Payne' – developed by Remedy Entertainment in 2001 and 2003 respectively – have a very distinct style of telling the story through a graphic novel. Panel by panel, the story plays out in still images (that are actually photographs of actors edited with filters to look more rough and sketchy) and we hear the voices of the characters. James McCaffrey provides the very distinct and grizzled voice for our titular NYPD detective, and delivers a fantastic performance in the process. The narration that he provides throughout the two games is superbly written to recreate that classic noir style of story-telling. The gameplay itself is very intentional in its homage to John Woo's cult classic "Hard Boiled" with the use of bullet-time, jumping and dodging in slow motion, giving you time to take out mobsters and look good doing it. The third game, 'Max Payne 3', was developed by Rockstar Studios and strayed from the relatively cheap but effective comic-book cutscenes. Although the visual aesthetic changed there, the characters, gameplay and level design (especially for the flashbacks in New York) are present and better than ever.
"There are no choices. Nothing but a straight line. The illusion comes afterwards, when you ask 'Why me?' and 'What if?' when you look back, see the branches, like a pruned bonsai tree, or a forked lightning."
This isn't a case of style over substance however, the story through all three titles is not only engaging but incredibly thought-provoking. The life of Max Payne is full of just that, maximum pain. Ultimately, the first game is a story of revenge – the murder of his wife and baby by druggies on a new fad called Valkyr – and through the excellent use of dream sequences, enacting how the situation would play on the characters mind, there is actually a layer of slight horror in some levels. The emotional impact is very strong, in between the exploring and shooting of NYC thugs.
"The genius of the hole: no matter how long you spend climbing out, you can still fall back down in an instant."
The sequel, set a few years after the original title, still keeps much of the thriller and action concepts from its predecessor but also intertwines a surprising love story as well. We follow Max trying to readjust to life in the NYPD, while people from his past make sudden cameos in his new cases, thus bringing him back into turmoil with the likes of many people, including Mona Sax, Vladimir Lem and the Inner Circle – a secret society we are introduced to in the latter part of the first game. We also play as more than just Max in this game for, even if it is just for a few chapters, which heightens both the gameplay and the narrative. Personally, 'The Fall of Max Payne' is actually the PEAK of Max Payne, as it's my favourite of the entire trilogy, and honestly one of my Top 5 games of all time. I find the story telling, the voice acting, the gameplay, the themes, the nuanced world building to be some of the best in any game I've played – and I would happily sit down now, to play it all again.
"When entertainment turns into a surreal reflection of your life, you're a lucky man if you can laugh at the joke. Luck and I weren't on speaking terms."
For quite some time, that was it. It was just two games. Until 2012 with the release of Max Payne 3, now set in São Paulo of all places. Max is now retired from the NYPD working as private security in Brazil. This is where the gameplay element was at it's finest. The bullet-time aspect, the general firefights, it was immaculately executed and was the genuine modern, next generation experience that Max Payne fans needed. The writing was still there too, with Max giving us more of his depressing narration, complete metaphors, similes and analogies. The visual aesthetic of the cutscenes, this time being fully animated CGI, wasn't quite as memorable or stylistic as the first two games, but it was distinctive enough in the way it would use typography, which certainly added gravitas to Max's impeccable quotes.
"In the land of the blind, a one eyed man is king."
Speaking of quotes, I've been dotting them through-out this write up. My word, if Max Payne wasn't a detective, he could most certainly be a poet. The way McCaffrey delivers the lines in a serious, monotone yet cool way is effortlessly badass, so couple that with wonderful writing from Sam Lake and you have lines of dialogue or narration that will stick in your mind for years to come. Sometimes it's on the border of cheesy, which is fitting for the action packed moments, but then it hits you again with a very serious (and sometimes relatable) quote about love/mental wellbeing/depression/mistakes/life and you're back to feeling engrossed in the media again, and likely feeling sorry for Max, with his alcoholism and painkiller addiction. Now, let's shoot some mobsters or drug dealers to make ourselves feel better.
"The way I see it there's two types of people, those who spend their lives trying to build a future and those who spend their lives trying to rebuild the past. For too long I'd been stuck in between, hidden in the dark."
So, yes, if you have yet to experience Max Payne, change that immediately. Unless it's the Mark Wahlberg movie. No-one needs to see that.
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Metabolic engineering is a commonly used approach to develop organisms for an industrial function, but engineering aimed at improving one phenotype can negatively impact other phenotypes. This lack of robustness can prove problematic. Cellulolytic bacterium Clostridium thermocellum is able to rapidly ferment cellulose to ethanol and other products. Recently, genes involved in H2 production, including the hydrogenase maturase hydG and NiFe hydrogenase ech, were deleted from the chromosome of C. thermocellum. While ethanol yield increased, the growth rate of ΔhydG decreased substantially compared to wild type.
Addition of 5 mM acetate to the growth medium improved the growth rate in C. thermocellum ∆hydG, whereas wild type remained unaffected. Transcriptomic analysis of the wild type showed essentially no response to the addition of acetate. However, in C. thermocellum ΔhydG, 204 and 56 genes were significantly differentially regulated relative to wild type in the absence and presence of acetate, respectively. Genes, Clo1313_0108-0125, which are predicted to encode a sulfate transport system and sulfate assimilatory pathway, were drastically upregulated in C. thermocellum ΔhydG in the presence of added acetate. A similar pattern was seen with proteomics. Further physiological characterization demonstrated an increase in sulfide synthesis and elimination of cysteine consumption in C. thermocellum ΔhydG. Clostridium thermocellum ΔhydGΔech had a higher growth rate than ΔhydG in the absence of added acetate, and a similar but less pronounced transcriptional and physiological effect was seen in this strain upon addition of acetate.
Sulfur metabolism is perturbed in C. thermocellum ΔhydG strains, likely to increase flux through sulfate reduction to act either as an electron sink to balance redox reactions or to offset an unknown deficiency in sulfur assimilation.
Microbial conversion of lignocellulosic biomass to fuels, chemicals, and products holds promise as a sustainable approach to replacing ones derived from petroleum. While no known organism is capable of producing cellulosic biofuels at high yield and titer, metabolic engineering has the potential to create biocatalysts capable of economic production of biofuels. However, engineered organisms often have growth defects, and this lack of robustness would prevent industrial deployment.
Clostridium thermocellum is a thermophilic, cellulolytic bacterium that has the potential to perform one-step hydrolysis and fermentation of plant biomass without added enzymes by a process called consolidated bioprocessing (CBP) . However, wild-type C. thermocellum is limited by its low ethanol yield and titer, producing acetate, lactate, H2, formate, free amino acids, and other compounds as additional fermentation products [2, 3]. Recent development of genetic tools for C. thermocellum [4–9] has enabled the construction of numerous targeted mutants, eliminating acetate, lactate, formate, and H2 production [4, 7, 10–14]. One of the mutations, C. thermocellum ΔhydG, eliminated a hydrogenase maturase protein involved in assembly of the [FeFe]-active site of three of the four hydrogenases in C. thermocellum. This deletion resulted in decreased H2 production and increased ethanol yield, but also a diminished growth rate. Further deletion of the [NiFe] hydrogenase Ech completely abolished H2 production . Because blocking H2 production alters electron flux, it is possible that this mutation causes a redox imbalance, which could also explain the slower growth. Further supporting this hypothesis, the ∆hydG mutant acquired a point mutation in the bifunctional aldehyde/alcohol dehydrogenase (adhE). This mutation expanded the substrate specificity to include not only NADH as an electron donor, but also NADPH , which could partially alleviate redox problems by giving the cell more pathways to balance redox reactions.
Understanding additional mechanisms used by C. thermocellum to balance redox reactions will be important for future metabolic engineering efforts. Here, we found that the addition of acetate to the culture medium increased the growth rate of C. thermocellum ΔhydG. We, therefore, utilized a combination of transcriptomics, proteomics, and physiological characterization to better understand this phenomenon.
Clostridium thermocellum DSM1313 and mutant strains C. thermocellum Δhpt ΔhydG (referred to as ΔhydG) and C. thermocellum Δhpt ΔhydG Δech (referred to as ΔhydG Δech)  were grown in CTFUD medium  and MTC minimal medium  prepared as described in . CTFUD medium composition was (L−1): 3 g sodium citrate tribasic dehydrate, 1.3 g ammonium sulfate, 1.43 g potassium phosphate monobasic, 1.8 g potassium phosphate dibasic trihydrate, 0.5 g cysteine-HCl, 10.5 g 3-morpholino-propane-1-sulfonic acid (MOPS), 6 g glycerol-2-phosphate disodium, 5 g cellobiose, 4.5 g yeast extract, 0.13 g calcium chloride dehydrate, 2.6 g magnesium chloride hexahydrate, 0.0011 g ferrous sulfate heptahydrate, and 0.0001 g resazurin, adjusted to pH 7.0. MTC medium consisted of (L−1): 2 g sodium citrate dehydrate, 1.25 g citric acid monohydrate, 1 g sodium sulfate, 1 g potassium phosphate dibasic trihydrate, 2.5 g sodium bicarbonate, 1.5 g ammonium chloride, 2 g urea, 1 g magnesium chloride hexahydrate, 0.2 g calcium chloride dehydrate, 0.1 g ferrous chloride tetrahydrate, 1 g l-cysteine hydrochloride monohydrate, 5 g cellobiose, 0.001 g resazurin, 5 g MOPS, 20 mg pyridoxamine dihydrochloride, 1 mg riboflavin, 1 mg nicotinamide, 0.5 mg DL-thioctic acid, 4 mg 4-amino benzoic acid, 4 mg D-biotin, 0.025 mg folic acid, 2 mg cyanocobalamin, 4 mg thiamine hydrochloride, 0.5 mg MnCl2·4H2O, 0.5 mg CoCl2·6H2O, 0.2 mg ZnSO4·7H2O, 0.05 mg CuSO4·5H2O, 0.05 mg HBO3, 0.05 mg Na2MoO4·2H2O, and 0.05 mg NiCl2·6H2O.
Genome resequencing was performed by the Department of Energy Joint Genome Institute (JGI, Walnut Creek, CA) using an Illumina MiSeq instrument. Genomic DNA was extracted using a Qiagen DNeasy kit (Qiagen, Valencia, CA), was sheared to 500 bp fragments using the Covaris LE220 ultrasonicator (Covaris), and size selected using AMPure XP SPRI beads (Beckman Coulter). The fragments were treated with end-repair, A-tailing, and ligation of Illumina compatible adapters (IDT, Inc) using the KAPA-Illumina library creation kit (KAPA Biosystems). The prepared libraries were quantified using KAPA Biosystem's next-generation sequencing library qPCR kit and run on a Roche LightCycler 480 real-time PCR instrument. The quantified multiplexed libraries were pooled in sets of 10, and sequenced on the Illumina MiSeq sequencer using an indexed PE150 protocol with MiSeq V2 chemistry.
Resequencing data analysis was performed using QIAGEN Bioinformatics CLC Genomics Workbench (http://www.qiagenbioinformatics.com/products/clc-genomics-workbench), which incorporates a comprehensive set of analysis tools for Next-Generation Sequencing data. Paired-end reads were mapped to the reference genome [Genbank: CP002416] using the built-in Map Reads to Reference Tool. Further refinement of the reads mapping was performed by the Local Realignment Tool, which attempts to re-align each mapped read by exploiting the alignment information of other mapped reads. Realignment typically occurs in areas around insertions and deletions in the sample reads relative to the reference, resulting in more accurate mapping. Mapped reads were next analyzed by the built-in tools Basic Variant Detection Tool for putative SNV and MNV detection, and InDels and Structural Variants Tool for detection of putative structural variants. Variants occurring in <90% of the reads and variants that were identical to those of the parent Δhpt strain (e.g., due to errors in the reference sequence or mutations present at the beginning of strain construction) were filtered out. Raw data are available from the JGI Sequence Read Archive (JGI Project Id: 1053867 and 1053888).
The inoculum for batch fermentation was prepared by growing the mutants in MTC medium overnight at 55 °C in an anaerobic chamber (COY Laboratory Products, Grass Lake, MI). The fermentation was carbon limited and carried out in 27 mL Balch tubes with 10 mL of MTC medium containing 5 g L−1 of cellobiose as the carbon source, supplemented with 5 mM sodium acetate where noted, under a N2 headspace sealed with butyl rubber stoppers. The tubes were inoculated with 0.5% v/v culture and incubated at 55 °C. The fermentation products were determined after 53 h of growth. Final cellobiose concentration was usually <0.5 mM, suggesting that fermentation activity was complete. Fermentations were performed at least two times with three independent biological replicates each. The "No Acetate" data were previously reported , which were generated simultaneously with the "Added Acetate" data reported here.
Fermentation products, including ethanol, acetate, lactate, and formate, were analyzed on Breeze 2 High-Performance Liquid Chromatograph system (Waters Corp, Milford, MA) using an Aminex-HPX-87H column with a 5 mM sulfuric acid mobile phase. Sulfide was measured using an Orion silver ion selective electrode (Thermo Fisher Scientific, Waltham, MA) as previously described . H2 was measured using a 6850 Series II Gas Chromatograph (Agilent Technologies, Santa Clara, CA) using a thermal conductivity detector at 190 °C with a N2 reference flow and a Carboxen 1010 PLOT (30.0 m × 530 µm I.D.; model Supelco 25467) column.
The cells were grown to an OD of 0.3–0.4 in CTFUD medium, centrifuged at 4 °C for 5 min, and immediately flash frozen in liquid N2. Pelleted cells were resuspended in 1.5 mL of TRIzol (Invitrogen, Carlsbad, CA). Glass beads (0.8 g of 0.1 mm glass beads; BioSpec Products, Bartlesville, OK) were added to the cell suspension and lysed with 3 × 20 s bead beating treatments at 6500 rpm in a Precellys 24 high-throughput tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA was purified using an RNeasy kit (Qiagen, Valencia, CA) with DNase I on-column treatment. RNA quantity was determined by NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and RNA quality was assessed with Agilent Bioanalyzer (Agilent Technologies). RNA (10 µg) was used as the template to generate ds-cDNA using Invitrogen ds-cDNA synthesis kit according to the manufacturer's protocols (Invitrogen).
The ds-cDNA was labeled, hybridized, and washed according to the NimbleGen protocols. Hybridizations were conducted using a 12-bay hybridization station (BioMicro Systems, Salt Lake City, UT) and the arrays dried using a Maui wash system (BioMicro Systems). Microarrays were scanned with a Surescan high-resolution DNA microarray scanner (5 µm) (Agilent Technologies), and the images were quantified using the NimbleScan software (Roche NimbleGen, Madison, WI). Raw data were log2 transformed and imported into the statistical analysis software JMP Genomics 6.0 (SAS Institute, Cary, NC). The data were normalized together using a single round of the LOESS normalization algorithm within JMP Genomics, and distribution analyses were conducted before and after normalization as a quality control step. An ANOVA was performed in JMP Genomics to determine differential expression levels between conditions using the False Discovery Rate (FDR) testing method (p < 0.05). Microarray data have been deposited in NCBI Gene Expression Omnibus (GEO) database under accession number (GSE54082). Data are average of three independent biological replicates.
Microarray data were validated using real-time qPCR, as described previously . Based on microarray hybridizations of C. thermocellum mutants, a set of 5 genes (Clo1313_0115, Clo1313_0147, Clo1313_0372, Clo1313_1559, and Clo1313_2243) representing a range of gene expression values was analyzed using qPCR from cDNA prepared for microarrays. Oligonucleotide sequences of the primers targeting the five genes selected for qPCR analysis are shown in Additional file 1: Table S1. Data are average of three independent biological replicates.
Crude protein fraction of C. thermocellum cell pellet was processed and digested with trypsin, and peptides were eluted and analyzed over an 11-step MudPIT as described previously . High mass accuracy was utilized for both MS1 (30 K resolution) and MS2 (7.5 K resolution; CID) scans (1 microscan each), with data-dependent acquisition settings as follows: 1 full scan followed by 20 MS/MS scans, isolation window = 2.1 m/z, dynamic exclusion window, duration, and max = −0.52/+ 1.02 m/z, 15 s, and 500, respectively. Peptides generated from C. thermocellum strain DSM1313 FASTA database concatenated with common contaminants and reversed entries were matched to MS/MS spectra using MyriMatch v. 2.1 . Common sample prep-induced modifications, i.e., Cys + 57.0214 Da (alkylation; static), Met + 15.9949 Da (oxidation; dynamic), and N-terminus + 43.0058 Da (carbamylation; dynamic), were included in the search parameters. Matches were filtered and assembled using IDPicker v. 3.0  using a minimum of two distinct peptides per protein identification and adjusting the minimum spectra count (SpC) per protein to achieve protein level FDRs < 5%, peptide-level FDRs < 1%, and PSM-level FDRs < 0.25%. Protein identifications with associated spectral counts (SpC) were tabulated, balanced, and normalized for semi-quantitative proteomics as previously described . Normalized SpC (nSpC) were used as a proxy for protein abundance across individual samples. To assess differences in protein abundance, the top 99% of total assigned spectra (across all sample conditions) was log2-transformed and processed by ANOVA (JMP Genomics v. 4.1) to assess statistical significance. Proteins with significant differences in abundance (p value ≤0.01) and minimum of twofold change were identified and compared with transcriptomics data to identify proteins affected by the knock-out of hydrogenases as well as the addition of acetate to the culture. Data are average of three independent biological replicates. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD000777.
Over the course of strain construction, we observed that growth of the C. thermocellum ΔhydG mutant was improved by the addition of exogenous acetate. Acetate was initially added based on the hypothesis that the addition of acetate could decrease flux through the acetate formation pathway , which could help prevent redox imbalances in the absence of H2 production. Indeed, acetate was a component of the culture medium during C. thermocellum ΔhydG Δech strain construction, and we were only successful in deleting ech in C. thermocellum ΔhydG when the medium contained added acetate. To characterize this growth phenotype, wild type, ΔhydG and ΔhydG Δech were grown in medium supplemented with 5 mM acetate. Growth of the wild-type strain was unaffected by the addition of acetate (Fig. 1a; 0.26 and 0.3 h−1, respectively, with nearly perfectly overlapping growth curves). However, in the presence of added acetate, the ∆hydG strain grew 33% more rapidly (0.16 h−1) than without added acetate (0.12 h−1) and attained a maximum optical density of 0.76 within 20 h (Fig. 1b), as opposed to 30 h in the absence of added acetate. While less pronounced, addition of acetate to the growth medium increased the growth rate of ∆hydG ∆ech as well, from 0.22 to 0.24 h−1 (Fig. 1c). While the growth rate was improved, the fermentation product profile did not substantially change upon addition of acetate (Table 1), with an ethanol yield similar to that reported previously without added acetate . Individual amino-acid concentrations are presented in Additional file 2: Figure S2 and are not substantially different from .
To balance redox reactions, each molecule of synthesized acetate requires the concomitant production of two molecules of a more reduced compound (e.g., formate or H2). Furthermore, there is a net production of NAD(P)H during microbial biomass formation from sugars . Thus, one would expect more formate and H2 to be produced than would be needed to balance the production of acetate alone. In wild type, 2 mM extra (formate + H2) was synthesized relative to the amount that would be required to balance acetate production (Additional file 1: Table S2). The mutant strains, on the other hand, produced approximately zero additional (formate + H2) beyond what is needed to balance acetate production, raising the possibility that an additional electron sink could be in use.
To gain insight into the cellular response to elimination of H2 formation and the addition of acetate, we explored changes in gene expression (Fig. 2; Additional file 3: Data set S1) and protein abundance (Additional file 4: Data set S2). A broad range of these gene expressions were also validated by RT-qPCR and a correlation coefficient of R 2 = 0.98 between microarray and qPCR analysis confirmed differential expression (Additional file 2: Fig. S3). In the absence of added acetate, 204 genes were significantly differentially expressed in ΔhydG compared to wild type, including 79 increased and 125 decreased in ΔhydG (Additional file 5: Data set S3). Genes with increased expression in ΔhydG included genes in Clusters 7 and 8 (Fig. 2b) associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) functions (Clo1313_2705-2713; Clo1313_2970-2976), glutamate biosynthesis (Clo1313_2035-2036), and cysteine metabolism (Clo1313_2325-2331). Genes with lowered expression included genes in Cluster 6 associated with sulfate uptake and assimilation (Clo1313_0115-0125) , and siroheme synthesis (Clo1313_0372-0375).
Surprisingly, addition of acetate to the fermentation medium had almost no effect on the transcriptomic profile of the wild-type strain, with only three genes significantly down regulated, including one iron sulfur cluster protein and two hypothetical genes (Fig. 2a), none of which were detected by proteomics (see below). Upon addition of acetate to the growth medium of ΔhydG, expression of 51 genes increased and five genes decreased. The two genomic regions of Cluster 6 in particular, consisting of total 27 genes, displayed dramatic increase in expression in the presence of added acetate. Proximal genes to these genomic loci were also upregulated in the presence of acetate and grouped in Cluster 1 (Fig. 2c). The first region from Clo1313_0108-0125 includes a predicted sulfate transport system and a complete sulfate assimilatory pathway, including genes for siroheme biosynthesis, which is a cofactor for sulfite reductase. The second genomic loci Clo1313_0372-0393 also included subunits of a putative pyruvate ferredoxin oxidoreductase, a putative carbon monoxide dehydrogenase, and two ABC transporters. Addition of acetate to ΔhydG Δech increased expression of cluster 6 as well, though not to the same degree as ΔhydG, since the basal (i.e., no added acetate) level of cluster 6 was not significantly different from wild type.
The upregulation of the Clo1313_0108-0125 and Clo1313_0372-0393 gene clusters in ΔhydG was further confirmed by proteomics using the same cultures harvested for transcriptomic analysis. About 1700 proteins were identified, out of which 21 were significantly upregulated and 37 downregulated in ΔhydG upon addition of acetate. Similar to the transcriptomics, acetate addition significantly increased the abundance of proteins from two clusters in ΔhydG strain: Clo1313_0110-0125 increased 78—709-fold and Clo1313_0373-0391 increased 2.7—408-fold (Additional file 4: Data set S2). Taken together, these data suggest that sulfur metabolism may be perturbed in C. thermocellum ΔhydG.
We also considered the possibility that differential protein acetylation could account for the difference in gene expression, but no differences in acetylation were detected in the proteomics data set (Additional file 4: Data set S4). The average number of detected acetylation events per sample was comparable between wild type (172), wild type with added acetate (155), ΔhydG (151), and ΔhydG with added acetate (152). Furthermore, no individual protein had statistically significant differences in acetylation.
With the increased abundance of transcripts and proteins related to sulfur metabolism, we examined the effect of deletion of hydG on sulfate reduction and cysteine metabolism. The measurement of sulfate consumption from the medium was masked by excess sulfate ions produced by the thermal degradation of the MOPS buffer during incubation at 55 °C during fermentation (Additional file 2: Fig. S3). However, sulfate is reduced to sulfide prior to incorporation into cysteine. While the wild type released ~7.5 μM sulfide into the culture medium, ~18 μM sulfide was released by ΔhydG, with similar results when acetate was added to the medium (Fig. 3a). As C. thermocellum is known to divert a significant flux of carbon and electrons toward production of secreted amino acids, we further examined the abundance of amino acids in the supernatant. Cysteine is provided in the culture medium as a reductant to make the medium anaerobic. Interestingly, the wild-type strain removed ca. 1.6 mM of 2.4 mM cysteine in the medium. C. thermocellum ΔhydG and ΔhydGΔech, on the other hand, did not consume any cysteine. Acetate addition in these mutants did not substantially alter uptake of cysteine from the medium for any of the strains (Fig. 3b).
The improved growth of C. thermocellum ΔhydGΔech, even in the absence of added acetate, was surprising given that metabolism is more constrained by the deletion of the Ech hydrogenase. We, therefore, considered the possibility that additional, beneficial mutations occurred during strain construction, and that these hypothesized mutations could be influencing the phenotype. To address this, we resequenced the genomes of C. thermocellum ΔhydG and ΔhydGΔech (Additional file 1: Table S3). The parent Δhpt strain contained 27 mutations relative to the published genome sequence, which likely represents a combination of mutations that have accumulated during and since strain construction as well as possible errors in the published genome. The expected deletions in hydG and ech were identified in the respective strains, as was the previously identified adhE mutation. Nine additional mutations were identified in ΔhydG relative to the parent strain. In ΔhydGΔech, four additional mutations were identified relative to ΔhydG. Of these ΔhydGΔech mutations, two result in amino-acid changes, and both could be related to gene expression: a putative ribonuclease and a putative transcription factor.
Understanding the mechanisms by which growth of mutated microbes can improve is important for understanding and improving strain robustness. While the hydrogenase mutants are some of the highest ethanol yielding C. thermocellum strains, the slow growth phenotype would likely be prohibitive for commercialization. By combining transcriptomics, proteomics, and physiological characterization, we have shed light on a mechanism by which growth of this mutant can be improved. Furthermore, acetate is abundant in many types of pant biomass in the form of acetylated xylan. Thus, hemicellulose-derived acetate could act as a growth stimulant for this strain.
Production of acetic acid + CO2 from sugars is obligately coupled to production of a more reduced compound, typically either H2 or formate in C. thermocellum, to balance redox reactions. Therefore, decreasing or eliminating H2 production without altering acetate production would result in a redox imbalance and decreasing flux through this pathway could help alleviate this imbalance. Thus, one might have expected that the addition of exogenous acetate could improve growth by lowering flux through the acetate production pathway. However, fermentation product profiles did not change substantially upon the addition of acetate. There was no net acetate consumption and strains are capable of growth without added acetate, demonstrating that deletion of hydG does not result in acetate auxotrophy. Instead, the addition of acetate to the culture medium increases the growth rate by altering sulfur metabolism (Fig. 4).
Sulfur metabolism is perturbed in C. thermocellum ΔhydG at the level of transcripts (Fig. 2), proteins (Additional file 4: Data set S2), and metabolites (Fig. 3). Amino-acid analysis demonstrated that ΔhydG strains do not consume cysteine from the medium and, therefore, must synthesize their own. One possible explanation is that deletion of hydG somehow blocks cysteine uptake through an unknown mechanism and thus requires the cell to make its own cysteine. Alternatively, a redox imbalance created by ΔhydG could be partially alleviated via sulfate reduction, which consumes four pairs of electrons per sulfate reduced to sulfide (Fig. 4). Wild type C. thermocellum consumed approximately 1.6 mM cysteine from the medium. If the hydrogenase mutants produce a similar amount of cysteine to fulfill cellular needs, then the necessary sulfate reduction would consume an amount of electrons equivalent to 6.4 mM H2 or formate. The combination of reduced acetate production relative to the wild type and increased sulfate reduction is likely sufficient to adequately balance redox reactions.
Hydrogenases are important for regenerating the oxidized ferredoxin used as the electron acceptor for Pyruvate:Ferredoxin Oxidoreductase (PFOR) during conversion of pyruvate to acetyl-CoA (Fig. 4). The sulfite reductase is predicted to utilize ferredoxin as the electron donor, which could help reoxidize the reduced ferredoxin generated by PFOR. Unlike sulfate-reducing bacteria, such as Desulfovibrio sp., where sulfate is used as the terminal electron acceptor in an energy-conserving electron transport chain [25, 26], in C. thermocellum, it is presumably an ATP-intensive pathway for cofactor re-oxidation. Although metabolically expensive, reduction of sulfate appears beneficial, as evident from the improved growth rate when this pathway is up regulated. Future efforts to provide alternate mechanisms to balance redox reactions have the potential to improve growth rate and also increase ethanol yield.
The role of the point mutation in the bifunctional alcohol/aldehyde dehydrogenase AdhE in the intersection between sulfur metabolism and redox balancing is unclear. With the expanded substrate specificity of AdhE, being able to use either NADH or NADPH for reduction of acetaldehyde to ethanol, the ΔhydG and ΔhydGΔech strains have an expanded range of possible pathways for redox balancing. However, C. thermocellum has a multitude of enzymes to enable the transfer of electrons between cofactors. Electrons can be directly transferred from ferredoxin to generate NADH using Rnf , while electrons from ferredoxin and NADH together can be used to reduce two NADP+ to NADPH [28, 29]. Furthermore, a carbon pathway, the malate shunt [30, 31], can functionally result in transhydrogenation during the conversion of phosphoenolpyruvate to pyruvate by oxidation of NADH by malate dehydrogenase and reduction of NADP+ by malic enzyme. The ability to utilize NADPH as a reductant for ethanol production would presumably provide additional flexibility in redox balancing, but the fact that sulfite reduction to sulfide is predicted to be ferredoxin-dependent makes the relevance of the mutant AdhE for sulfur metabolism tenuous.
The increased flux through sulfate reduction appears to occur both with and without added acetate, as evidenced by the lack of cysteine consumption in each case. Thus, the level of proteins involved in sulfate reduction appears to be rate limiting. The addition of acetate increases expression of the necessary genes, increasing the rate of sulfate reduction and, thus, the rate of growth. This also explains the smaller impact of acetate addition in ΔhydGΔech, where the expression level of this gene cluster is already at a higher basal level in the absence of added acetate. The higher basal expression presumably permits higher flux through sulfate reduction, even in the absence of added acetate. Interestingly, two non-synonymous mutations were identified in ΔhydGΔech in a putative ribonuclease and a transcription factor. It is conceivable that one or both of these mutations alter either mRNA stability or transcription, respectively, to return gene expression levels without added acetate to wild-type levels for gene cluster 6. Furthermore, sulfide is a volatile intermediate in the production of cysteine, so the wild-type level of sulfide production in ΔhydGΔech could indicate efficient assimilation of the synthesized sulfide in this strain.
Addition of acetate to wild-type cultures had a little impact, which is not surprising, because the level of added acetate (5 mM) is substantially lower than the amount wild-type strain produces during growth on 5 g/L cellobiose (approximately 9 mM under these conditions; see Table 1). However, supplementation with acetate modulates gene expression and metabolic flux in C. thermocellum ΔhydG by an unknown mechanism. Because of the role of hydrogenases in reoxidizing ferredoxin, deletion of hydG could reasonably affect flux through PFOR to perturb pools of acetyl-CoA and acetyl-phosphate, which are known to acetylate proteins to alter enzymatic properties in some organisms [32, 33]. However, no difference in protein acetylation was observed in ΔhydG either in the presence or absence of added acetate, suggesting that differential protein acetylation is not the likely mechanism by which sulfate reduction genes are more highly expressed. Thus, the mechanism of gene upregulation remains to be elucidated.
This transcriptomic response of ΔhydG to the addition of acetate is similar to that seen in wild type C. thermocellum upon exposure to furfural , a toxic aldehyde created by the dehydration of xylose at high temperature, such as during plant biomass pretreatment. In organisms like E. coli, furfural is detoxified via NADPH-dependent reduction to furfuryl alcohol, which is less toxic. This is thought to decrease the amount of NADPH available for biosynthesis and inhibit growth . Supplementation of E. coli with cysteine or increasing the concentration of NADPH increased furfural tolerance, suggesting that cysteine metabolism is intimately tied to the redox state of the cell. Sulfate reduction genes are also differentially expressed in response to chemical redox perturbation , and addition of excess sulfate helped recover viability of a C. thermocellum mutant with perturbed redox metabolism . How sulfur metabolism relates to redox homeostasis requires further study and may inform future metabolic engineering strategies.
Understanding and overcoming low robustness in engineered microorganisms will be essential to industrial deployment of these organisms. The previous deletion of hydrogenases in C. thermocellum resulted in strains with a higher ethanol yield but a substantially lower growth rate. Here, we find that this growth defect can be overcome with the addition of acetate to the medium, which results in an increase in sulfate reduction. Sulfate likely serves as an electron acceptor to help balance redox reactions, which has implications for future metabolic engineering efforts.
RB participated in the design of the study, characterized growth and fermentation, grew cultures for –omics analyses, and drafted the manuscript. CMW, DMK, and SDB performed and analyzed transcriptomics experiments. RJG and RLH performed and analyzed proteomics experiments. TR and MBS analyzed and interpreted genome resequencing data. AMG helped conceive of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.
We would like to acknowledge Daniel G. Olson for his assistance with resequencing data analysis and helpful discussions. This work was supported by the BioEnergy Science Center, U.S. DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725.
The microarray data generated during and/or analyzed during the current study are available in the NCBI Gene Expression Omnibus (GEO) database under accession number (GSE54082). The proteomics data generated during and/or analyzed during the current study are available in the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD000777. All other data generated or analyzed during this study are included in this published article and its supplementary information files.
This work was supported by the BioEnergy Science Center, U.S. DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under Contract No. DE-AC02-05CH11231. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
13068_2016_684_MOESM1_ESM.xlsx Additional file 1: Supplemental Tables. Table S1. Primers used in this study. Table S2. Calculations of electron balance for strains with and without added acetate. Table S3. Analysis of mutations present in strains relative to the published wild type sequence.
13068_2016_684_MOESM2_ESM.docx Additional file 2: Supplemental Figures. Figure S1. Correlation plot between microarray and RT-qPCR data sets. Verification of differential gene expressions in C. thermocellum in the presence and absence of acetate. Figure S2. Concentration of amino acids produced by wild type, ΔhydG, and ΔhydGΔech strains with/out added acetate.*, asparagine quantification for two samples was prevented by interference. Error bars represent one standard deviation. Figure S3. Incubation of MTC medium at high temperature releases sulfate. Sulfate concentration in culture medium after incubation for 5 days. RT, room temperature; 55 °C, incubation at 55 °C; presence (black bars) and absence (gray bars) added acetate.
13068_2016_684_MOESM3_ESM.xlsx Additional file 3: Data set S1. Hierarchical clustering of gene expression. Genes significantly (p < 0.05) differentially expressed in various comparisons between the wild-type parental strain, the ΔhydG and ΔhydG Δech mutant C. thermocellum strains in the presence or absence of acetate. Expression differences are expressed as log2 transformed values. Yellow shading indicates upregulation by a fold change of 2 in first condition listed, while blue indicates downregulation of gene expression by a fold change of 2. For example: in the comparison, "WT (+acetate)–WT (−acetate)" yellow shading would indicate a significant upregulation of genes in the WT strain grown with acetate compared to the WT strain grown without acetate. Conversely, blue shading indicates higher expression when the cells were grown without acetate compared to those grown with acetate. Values in gray indicate the significance of the differential gene expression was below the p < 0.05 threshold for that specific comparison. Functional annotations are given of those available in the public databases. Values in the column Hierarchical cluster cross-reference to clusters in Fig. 4.
13068_2016_684_MOESM4_ESM.xlsx Additional file 4: Data set S2. Proteomic analysis of wild type and ΔhydG in the presence or absence of acetate. Proteomic analysis of wild type and ΔhydG in the presence or absence of acetate. Color code of the p values based on significance, with red indicating highly significant and white non-significant.
13068_2016_684_MOESM5_ESM.xlsx Additional file 5: Data set S3. Differential gene expression. Differential gene expression expressed as a ratio between the ΔhydG, ∆hydG ∆ech, and wild-type strains of C. thermocellum in the presence or absence of acetate. Included is the False Discovery Rate (FDR) adjusted p value for each gene comparison with an FDR adjusted p value <0.05 and greater than ±1 log2 transformed ratio between the conditions indicative of altered gene regulation.
13068_2016_684_MOESM6_ESM.xlsx Additional file 6: Data set S4. Acetylation analysis of ΔhydG and ∆hydG∆ech strains. Acetylation of the proteins in mutants were compared to wild type in the presence or absence of acetate.


text:
Q: running sample typeahead in bootstrap i just started using bootstrap and the following jsfiddle: https://jsfiddle.net/nt3qsjbx/ contains the sample code for typeahead.
I am not sure which this is not working!!
$('#country_v1-query').typeahead({
    order: "desc",
    source: {
        data: [
            "Afghanistan", "Albania", "Algeria", "Andorra", "Angola", "Antigua and Barbuda",
            "Argentina", "Armenia", "Australia", "Austria", "Azerbaijan", "Bahamas", "Bahrain", "Bangladesh",
            "Barbados", "Belarus", "Belgium", "Belize", "Benin", "Bermuda", "Bhutan", "Bolivia",
            "Bosnia and Herzegovina", "Botswana", "Brazil", "Brunei", "Bulgaria", "Burkina Faso", "Burma",
            "Burundi", "Cambodia", "Cameroon", "Canada", "Cape Verde", "Central African Republic", "Chad",
            "Chile", "China", "Colombia", "Comoros", "Congo, Democratic Republic", "Congo, Republic of the"... ]});

A: Have a look at Twitter Typeahead
It's quite easy to use and straight forward.
Here is a good example of how to use it: https://twitter.github.io/typeahead.js/examples/

redpajama_set_name: RedPajamaStackExchange


text:
Fantastic Fest Review: Come Clean With THE CLEANSING HOUR
The demons have joined the streaming war.
As we find ourselves amidst streaming wars between giants like Netflix, Disney+, YouTube, and the forty other services that have cropped up in the last three weeks, it's natural to wonder how those wars might escalate. The Cleansing Hour feels like the perfect demonic answer to that question. Reverend Max (Ryan Guzman), his best friend Drew (Kyle Gallner), and their devoted production team have a pretty good gig going on their exorcism show. But they find themselves hosts to some unexpected guests before their shoot wraps.
Their show, The Cleansing Hour, features the eye candy Reverend exorcising demon after demon for followers and merch sales. Max is wrapped up in the fame of it all, whining about his lack of Twitter verification and begrudging those with more of a following than him. On the other hand, Drew just wants to help the show expand and evolve into something new so they can grow their little production into something more. Both men learn that they should be careful what they wish for before the night's over.
The third and most impressive player in The Cleansing Hour is the show's makeup artist, Lane (Alix Angelis). While the most vocal in her distaste for Max's constant peacocking, she finds herself forced to step in when that night's possession no shows. They say that the show must go on, but she probably would've bowed out if she knew just what that would mean for her and fiancé, Drew.
When the three of them find themselves in a very real possession scenario, they're forced to either air their grievances and share their darkest confessions, or watch their friends die. It's at this point that things really shift to focus on Angelis' performance, which ends up being right in line with their reliance on practical effects whenever possible as the best decision of the movie.
The Cleansing Hour feels like the exact kind of horror flick to introduce folks to the genre. It's not the perfect film. There are a couple of rough CGI moments, and a few clichés slip their way into the plot. But, at the end of the day, I spent a lot of the movie grinning. The script is fresh, and everything wrapped together resulted in a genuinely fun take on exorcism and possession in the digital age. Fear Dot Com meets the snark of a Supernatural demon. It plays it straight and has a hell of a lot of fun while doing it.
Fear Dot Com
DVD | Warner Home Video
the cleansing hour