How to get nitrogen out of the air – Legume-rhizobia symbiosis

Nitrogen is an essential nutrient as it makes up many biochemical compounds. It is found in the nucleoside triphosphates and amino acids that form the building blocks of nucleic acids and proteins, respectively. Therefore all organisms must gain access to a supply of nitrogen in a form they can assimilate in order to survive.

Dinitrogen (N2) makes up roughly 78% of the atmosphere by volume [1]. However, despite its abundance, plants are unable to assimilate nitrogen in this form. Dinitrogen has a bond energy of 941.4 kJ mole-1 [2] which makes it an exceptionally stable molecule. Eukaryotes do not have the enzymatic capability to break the triple bond. Only a small number of prokaryotes, the nitrogen fixing bacteria, [1] have the ability to break the triple bond and fix the nitrogen into ammonium which plants are able to assimilate. Therefore the only way plants can gain access to atmospheric nitrogen is via symbiosis.

Nitrogen fixing bacteria, including rhizobia, account for 90% [1] of the naturally fixed nitrogen compounds in the soil. Without nitrogen fixing bacteria the soil would be unable to sustain plant population density anywhere near its present level. Rhizobia comprise gram negative soil bacteria of the genera Azorhizobium, Bradyrhizobium, Photorhizobium, Rhizobium, and Sinorhizobium [3]. Most nitrogen fixing bacteria are free-living, however a small but significant number are capable of forming symbiotic relationships with plants. The most common type of symbiosis occurs between plants such as beans, pea, lentils, clover, alfalfa, or vetch, (which are part of the family Leguminosae), and rhizobia [1]. Under nitrogen limited conditions the symbionts seek out one another through an elaborate exchange of signals. This signalling leads to a subsequent infection process involving Ca2+ concentration cytosolic spiking [4] and root hair curling culminating in the formation of an infection thread. Eventually mature root nodules containing bacteroids, nitrogen fixing rhizobia surrounded by a plant derived membrane, are formed. These nodules capture atmospheric nitrogen for the mutual benefit of the bacteria and plant.


  1. Taiz, L. & Zeiger, E. (2002) Assimilation of mineral nutrients. In: Plant Physiology. 3rd ed. A.D. Sinauer ed. pp. 260-272. Sinauer Associates, Inc.
  2. Chang, R. (2002) Chemical bonding I: basic concepts. In: Chemistry. 7th ed. K.A. Peterson ed. p. 356. McGraw-Hill.
  3. Buchanan, B.B., Gruissem, W. & Jones, R.L. (2000) Nitrogen and Sulfur. In: Biochemistry & Molecular Biology of Plants. N.M. Crawford, M.L. Khan, T. Leustek, & S.R. Long ed.s. pp. 796-797. The American Society of Plant Biologists.
  4. Oldroyd, G.E.D. & Downie, J.A. (2004) Calcium, kinases and nodulation signalling in legumes. Nat. Rev. Mol. Cell. Biol. 5:566-76.

CAVATAK oncolytic virus

CAVATAK is a proprietary Coxsackievirus A21 (CVA21) developed by Viralytics. CVA21 is a naturally occurring “common cold” RNA virus which targets the intercellular adhesion molecule-1 (ICAM-1). CAVATAK CVA21 is a novel bio-selected formulation, which has potent oncolytic activity against in vitro cultures of cancer cells and in vivo xenografts of a number of cancers. It has demonstrated phase II trial safety in late stage melanoma patients with evidence of induction of immune cell infiltration into the tumours [1]. Preclinical data in a mouse melanoma model suggest combinations of CAVATAK and anti-PD-1 or anti-CTLA-4 mAbs mediated significantly greater antitumor activity and offered greater survival benefits when compared to use of either agent alone [2].


  1. Andtbacka, R.H.I., Curti, B.D., Kaufman, H., Daniels, G.A., Nemunaitis, J.J., Spitler, L.E., Hallmeyer, S., Lutzky, J., Schultz, S.M., Whitman, E.D., et al. (2015). Final data from CALM: A phase II study of Coxsackievirus A21 (CVA21) oncolytic virus immunotherapy in patients with advanced melanoma. J. Clin. Oncol. 33,     suppl; abstr 9030.
  2. Au, G., Quah, M., Wong, Y., and Shafren, D. (2015). Combination of a novel oncolytic immunotherapeutic agent, CAVATAKTM (Coxsackievirus     A21) and immune-checkpoint blockade significantly reduces tumor growth and improves survival in an immune competent mouse melanoma model. In 9th International Conference on Oncolytic Virus Therapeutics, (Boston), p. O – 34.


ERK inhibitors – a valuable new tool for treating RAS/RAF active cancers

It has been found that in half of cell lines with RAS activation mutations the inhibition of ERK1/2 results in growth arrest including those that have acquired resistance to MEK inhibitors [1]. This is important as 50% of patients who are treated with BRAF or MEK inhibitors have disease progression within 6 to 7 months after the initiation of treatment [2].

An ERK1/2 inhibitor may prove effective in combination with MEK/BRAF inhibitors as a “double hit” of the MAPK pathway in RAS/RAF active cancers [3]. The double hit reduces the chances the tumours will spontaneously develop the mutations required to overcome inhibition of the pathway [4].

Novel ERK inhibitors such as Merck’s SCH772984/ MK-8353 or others may well be useful in MAPK combination therapy for melanoma and pancreatic cancer. It should be noted however that a phase I clinical trial of SCH772984/ MK-8353 was terminated by Merck without reason given be it financial or otherwise [5].


  1. Hayes, Tikvah K., Nicole F. Neel, Chaoxin Hu, Prson Gautam, Melissa Chenard, Brian Long, Meraj Aziz, et al. ‘Long-Term ERK Inhibition in KRAS-Mutant Pancreatic Cancer Is Associated with MYC Degradation and Senescence-like Growth Suppression’. Cancer Cell 29, no. 1 (January 2016): 75–89. doi:10.1016/j.ccell.2015.11.011.
  2. Flaherty, Keith T., Jeffery R. Infante, Adil Daud, Rene Gonzalez, Richard F. Kefford, Jeffrey Sosman, Omid Hamid, et al. ‘Combined BRAF and MEK Inhibition in Melanoma with BRAF V600 Mutations’. New England Journal of Medicine 367, no. 18 (November 2012): 1694–1703. doi:10.1056/NEJMoa1210093.
  3. Morris, E. J., S. Jha, C. R. Restaino, P. Dayananth, H. Zhu, A. Cooper, D. Carr, et al. ‘Discovery of a Novel ERK Inhibitor with Activity in Models of Acquired Resistance to BRAF and MEK Inhibitors’. Cancer Discovery 3, no. 7 (1 July 2013): 742–50. doi:10.1158/2159-8290.CD-13-0070.
  4. Hatzivassiliou, Georgia, Bonnie Liu, Carol O’Brien, Jill M. Spoerke, Klaus P. Hoeflich, Peter M. Haverty, Robert Soriano, et al. ‘ERK Inhibition Overcomes Acquired Resistance to MEK Inhibitors’. Molecular Cancer Therapeutics 11, no. 5 (5 January 2012): 1143–54. doi:10.1158/1535-7163.MCT-11-1010.
  5. ‘A Study of the Safety, Tolerability, and Efficacy of MK-8353 in Participants With Advanced Solid Tumors (MK-8353-001) – Full Text View –’. Accessed 27 January 2016.

Tumour lysis syndrome

Tumour lysis syndrome is a consequence of unusually high levels of tumour cell lysis over a short time period. It may be triggered by the initiation of cancer therapy. Laboratory tumour lysis syndrome is defined as hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia in response to therapy. Tumour lysis syndrome is unlikely without the previous development of nephropathy (kidney failure) and a consequent inability to excrete solutes quickly enough to cope with the metabolic load. Clinical tumour lysis syndrome also includes increased creatinine level, seizures, cardiac dysrhythmia, or death. Tumour lysis can also release cytokines that cause a systemic inflammatory response syndrome and often multi-organ failure [1].

Cancers with a high potential for cell lysis include high-grade lymphomas, acute leukaemias, and other rapidly proliferating tumours. During cancer drug development an important aspect of phase I trials is dose escalation (patients receive the same drug but some get a higher concentration than others over a relevant step range). This allows monitoring for safety issues associated with dose such as laboratory tumour lysis syndrome. An example of a drug in which tumour lysis syndrome was found to be an issue at certain doses was Venetoclax for treatment of relapsed chronic lymphocytic leukaemia (CLL) [2].

CLL is characterised by long lived lymphocytes that do not undergo apoptosis due their expressing high levels of BCL2. New lymphocytes are constantly generated under normal circumstances but in CLL without apoptosis to eliminate old lymphocytes load increases beyond normal levels. Venetoclax is a BH3 mimetic which binds BCL2 and releases pro-apoptotic signalling pathways from inhibition. In the phase I study clinical tumour lysis syndrome occurred in 3 of 56 patients in the dose-escalation cohort, with one death. After adjustments to the dose-escalation schedule, clinical tumour lysis syndrome did not occur in any of the 60 patients in the expansion cohort [2]. This highlights the risk of clinical tumour lysis syndrome in CLL patients treated with the highly effective Venetoclax. Some experts advise that in the clinic the Venetoclax dose should be increased in increments gradually for each individual patient with careful monitoring [3].


  1. Howard, Scott C., Deborah P. Jones, and Ching-Hon Pui. ‘The Tumor Lysis Syndrome’. New England Journal of Medicine 364, no. 19 (12 May 2011): 1844–54. doi:10.1056/NEJMra0904569.
  2. Roberts, Andrew W., Matthew S. Davids, John M. Pagel, Brad S. Kahl, Soham D. Puvvada, John F. Gerecitano, Thomas J. Kipps, et al. ‘Targeting BCL2 with Venetoclax in Relapsed Chronic Lymphocytic Leukemia’. New England Journal of Medicine, 6 December 2015, 151206090218007. doi:10.1056/NEJMoa1513257.
  3. ‘Episode 10: New Horizons in Hematology’. Novel Targets Podcast, 7 January 2016.

Teixobactin a much needed new antibiotic

At a time when multi-drug resistant bacteria are proliferating, antibiotic development by big pharma has taken a steep decline during the 90s and 2000s for a number of reasons. To address this regulatory and economic incentives have been put in place to encourage big pharma to once again invest in antibiotics. It has been predicted that if the problem of multi-drug resistant bacteria is not addressed immediately they will potentially cause the death of 300 million people during the next 35 years [1]. This is the antibiotic crisis.

Awareness is growing and there have been some very encouraging recent developments which should allow small scale biotech to take centre stage in antibiotic discovery. It may sound surprising but it is nonetheless true (and encouraging) that 99% of bacteria in the natural environment are uncultured [2]. New simple techniques have been developed that allow many of these “unculturable” bacteria to be grown.

A technology called the iChip facilitates high-throughput culture of soil samples. It was used in the discovery of a new species of ß-proteobacteria provisionally named Eleftheria terrae. E. terrae secretes an antibiotic called teixobactin which is effective against gram positive (no outer membrane) pathogens such as Staphylococcus aureus and Mycobacterium tuberculosis.

Teixobactin inhibits the synthesis of peptidoglycan which is gram positive bacteria’s main defense against environmental factors. In addition inhibiting peptidoglycan synthesis leads to the buildup of toxic intermediates that lyse gram positive bacteria [2].

There are many more antibiotics to be found in the soil.


  1. Bettiol, Esther, and Stephan Harbarth. ‘Development of New Antibiotics: Taking off Finally’. Swiss Med Wkly 145 (2015): w14167.
  2. Ling, Losee L., Tanja Schneider, Aaron J. Peoples, Amy L. Spoering, Ina Engels, Brian P. Conlon, Anna Mueller, et al. ‘A New Antibiotic Kills Pathogens without Detectable Resistance’. Nature 517, no. 7535 (7 January 2015): 455–59. doi:10.1038/nature14098.

TGF-β1 induced EMT

Epithelial to mesenchymal transition (EMT) is a process by which cells of an epithelial phenotype dedifferentiate to a motile mesenchymal phenotype. The Epithelial cell reorganizes its actin cytoskeleton from a cortical junctional associated arrangement to form motile stress fibres, redistributes and down regulates its cell-cell contacts, loses its polarity, and upregulates mesenchymal markers such as α-smooth muscle actin (α-SMA) and vimentin [1]. The cell also shifts its association with the basal lamina by altering the composition of its extracellular matrix (ECM) contacts and secreting matrix metalloproteinases [2]. EMT has a role during development [3], chronic fibrotic disorders [4], and a postulated role in epithelial cancer metastasis [5].

Transforming growth factor beta-1 (TGF-β1) can trigger an EMT in cancer cells leading to metastasis [5]. In order for an adenocarcinoma cell to metastasize and relocate to a different site, it must somehow lose contact with its surrounding cells, become motile and acquire the ability to manipulate the basal lamina in order to intravasate into local blood vessels or lymph system. An EMT triggered by TGF-β1 provides a hypothetical mechanism for this occurrence. It is considered important that cancerous cells lose sensitivity to the growth inhibitory effect of TGF-β1 before they undergo EMT in vivo [5]. However loss of growth inhibition is not necessarily correlated with EMT in vitro [6].

Although the phenotypic characteristics of EMT are fairly well defined, the signalling pathways responsible for TGF-β1 induced EMT are not. The mitogen activated protein kinases (MAPKs), extracellular signal regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) have all been implicated despite no known direct link between TGF-β receptors and MAPK pathways [7,8,9]. In addition several other pathways have been implicated including Notch signalling (which is modulated by TGF-β signalling), β-catenin signalling, and Akt signalling [10,11,12]. A critical step in understanding both EMT and TGF-β signalling will be deciphering how these pathways are activated in response to TGF-β.


  1. Savagner, P. 2001. Leaving the neighborhood: molecular mechanisms involved during Epithelial-Mesenchymal Transition. BioEssays. 23: 912-923.
  2. LaGamba, D. Nawshad, A. and Hay, E.D. 2005. Microarray analysis of gene expression during Epithelial-Mesenchymal Transformation. Dev Dyn. 234: 132-42
  3. Hay, E.D. 1995. An overview of Epithelio-Mesenchymal Transformation. Acta Anat (Basel). 154: 8-20.
  4. Kalluri, R. and Neilson, E.G. 2003. Epithelial-Mesenchymal Transition and its implications for fibrosis. J Clin Invest. 112: 1776-84.
  5. Thiery, J.P. 2002. Epithelial-Mesenchymal Transitions in tumour progression. Nat Rev Cancer. 2: 442–454.
  6. Brown, K.A. Aakre, M.E. Gorska, A.E. Price, J.O. Eltom, S.E. Pietenpol, J.A. and Moses, H.L. 2004. Induction by Transforming Growth Factor-β1 of Epithelial to Mesenchymal Transition is a rare event in vitro. Breast Cancer Res. 6: R215-R231
  7. Xie, L. Law, B.K. Chytil, A.M. Brown, K.A. Aakre, M.E. Moses, H.L. 2004. Activation of the Erk pathway is required for TGF-β1–induced EMT in vitro. Neoplasia. 6: 603-610
  8. Bakin, A.V. Rinehart, C. Tomlinson, A.K. Arteaga, C.L. 2002. p38 Mitogen-Activated Protein Kinase is required for TGF-β-mediated fibroblastic transdifferentiation and cell migration. J Cell Sci. 115: 3193-3206.
  9. Engel, M.E. McDonnell, M.A. Law, B.K. and Moses, H.L. 1999. Interdependent SMAD and JNK signaling in Transforming Growth Factor-β-mediated transcription. J Biol Chem. 274: 37413-37420.
  10. Zavadil, J. Cermak, L. Soto-Nieves, N. Böttinger, E.P. 2004. Integration of TGF-β/Smad and Jagged1/Notch signalling in Epithelial-to-Mesenchymal Transition. EMBO J. 23: 1155-1165.
  11. Kim, K. Lu, Z. and Hay, E.D. 2002. Direct evidence for a role of β-Catenin/LEF-1 signaling pathway in induction of EMT. Cell Biol Int. 26: 463–476.
  12. Bakin, A.V. Tomlinson, A.K. Bhowmick, N.A. Moses, H.L. and Arteaga, C.L. 2000. Phosphatidylinositol 3-Kinase function is required for Transforming Growth Factor β -mediated Epithelial to Mesenchymal Transition and cell migration. J Biol Chem. 275: 36803-36810.

The role of the tetraspanin CD9 in metastasis – A double edged sword?

The current consensus is that CD9 is a metastasis suppressor. CD9 expression has been inversely associated with the metastasis of cancers. That is low CD9 protein expression is associated with tumour samples taken from secondary metastatic sites compared to the primary tumour site. Furthermore in a number of cancer cell lines and primary cells, CD9 expression has been shown to decrease cell motility. A number of studies however conversely demonstrate that CD9 can increase the motility of cancer and “normal” cells. This does not fit with the metastasis suppressor hypothesis and has not been adequately explained by current models of CD9 function during metastasis.

These apparently contradictory observations may stem from the large number of functionally distinct steps that the term metastasis covers. … Furthermore recent evidence suggests that contrary to the traditional view that metastasis occurs late in cancer progression, metastasis may occur early in relatively normal cells. This has serious implications for the role of CD9 and many other proteins in metastasis. CD9 is widely expressed in many normal cells. If metastasis occurs early this suggests that CD9 expression does not inhibit metastasis.