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PHILADELPHIA, PA and REHOVOT, ISRAEL / ACCESSWIRE / May 5, 2017 / Rosetta Genomics Ltd. (NASDAQ: ROSG), a genomic diagnostics company that improves treatment decisions by providing timely and accurate diagnostic information to physicians, announces that the Company has signed an exclusive distribution agreement with Cytolog Laboratories (Cytolog) for the sales and marketing of RosettaGX RevealTM (Reveal), a first-of-its-kind microRNA assay for the classification of indeterminate thyroid nodules, in Brazil. Cytolog is a Brazilian-based diagnostics laboratory that specializes in fine needle aspiration examinations. Cytolog already has the regulatory approvals to send biological samples outside of Brazil and expects to begin utilizing RosettaGX Reveal effective immediately. "We are delighted to partner with Rosetta Genomics to bring RosettaGX Reveal to patients in Brazil who have an indeterminate thyroid cancer diagnosis. In addition to its excellent performance, Reveal has a significant advantage to current assays on the market because it can work off the same cytology slides that were created to perform the initial diagnosis, thus eliminating the risks, added patient stress, and unnecessary pain associated with additional fine needle passes," stated Fabiano Callegari, M.D., pathologist and principal of Cytolog. "We look forward to bringing Reveal to the Brazilian market as many patients with indeterminate thyroid cancer undergo surgery as a precaution despite the fact that up to 80% of these cases are benign. This exposes patients to unnecessary surgical risk and costs the healthcare system millions of dollars," added Dr. Callegari. According to Surveillance, Epidemiology & End Results (SEER), a U.S. National Cancer Institute program that collects incidence and survival data from cancer registries, the incidence of thyroid cancer has tripled in the past 35 years. In Brazil, the National Cancer Institute (INCA) estimated 9,200 new cases in 2014, 8,050 of them in women. This is largely the result of improved diagnosis due to the increased use of ultrasound scanning. "We are particularly pleased to expand access for Reveal to the Brazilian market where the incidence of thyroid cancer diagnosis is growing," stated Kenneth A. Berlin, President and Chief Executive Officer of Rosetta Genomics. "Unlike other assays in the space, Reveal does not require fresh tissue and therefore does not require special logistics for sample handling and shipment. These simple logistics make it ideally suited for international distribution agreements like this one. We look forward to working with Cytolog, a premier laboratory with a reputation as a leader in the field of fine needle aspirates. This is our first distribution agreement in South America and it marks a significant milestone in our strategic goal to expand global access for Reveal." Cytolog is a Brazilian-based laboratory that specializes in fine needle aspiration examinations, which today is a reference in precise diagnoses, equaled to the best medical centers in the world and certified by the Brazilian Societies of Pathology and Cytopathology. The evolution of medicine has been generous to patients, not only for seeking healing for their illnesses but also for providing an early diagnosis. Proof of this is the fine needle aspiration examination, which can replace the surgical biopsy, making the procedure quick, simple and well accepted by patients. This examination consists of the cytological analysis of thyroid nodules, salivary glands, breast, and lymph nodes, among other lesions. Cytolog has very strong human values and therefore offers personalized accompaniment to all its patients, providing much more comfort and well-being. Rosetta is pioneering the field of molecular diagnostics by offering rapid and accurate diagnostic information that enables physicians to make more timely and informed treatment decisions to improve patient care. Rosetta has developed a portfolio of unique diagnostic solutions for oncologists, urologists, endocrinologists, cytopathologists and other specialists to help them deliver better care to their patients. RosettaGX Reveal™, a Thyroid microRNA Classifier for the diagnosis of cancer in thyroid nodules, as well as the full RosettaGX™ portfolio of cancer testing services are commercially available through the Company's Philadelphia, PA- and Lake Forest, CA-based CAP-accredited, CLIA-certified labs. Various statements in this release concerning Rosetta's future expectations, plans and prospects including, but not limited to bringing Reveal to the Brazilian market and expanding global access and statements containing the words "expect," "believe," "will," "may," "should," "project," "estimate," "anticipated," "scheduled," and like expressions, and the negative thereof, constitute forward-looking statements for the purposes of the safe harbor provisions under The Private Securities Litigation Reform Act of 1995. Actual results may differ materially from those indicated by these forward-looking statements as a result of various risks and uncertainties, including, but are not limited to the following: we will require substantial additional funds to continue our operations and, if additional funds are not available, we may need to significantly scale back or cease our operations; we have a history of losses and may never be profitable; if we are unable to expand sales of our diagnostic tests in the United States, it would have a material adverse effect on our business and financial condition; the intensely competitive biotechnology market could diminish demand for our tests and products; the market may not be receptive to any diagnostic tests or therapeutic products using our microRNA technology; we currently have limited sales, marketing or distribution experience and may in the future depend significantly on third parties to commercialize microRNA-based diagnostic tests or therapeutic products we may develop; we are largely dependent upon our distributors for the success of commercialization of our current diagnostic tests; health insurers and other third-party payors may decide not to cover our diagnostic products or may provide inadequate reimbursement, which could jeopardize our commercial prospects; because of Medicare billing rules, we may not receive reimbursement for all tests provided to Medicare patients; if we fail to comply with our obligations under any licenses or related agreements, we could lose license rights that may be necessary for developing microRNA-based diagnostics and therapeutics; if we fail to comply with the complex federal, state, local and foreign laws and regulations that apply to our business, we could suffer severe consequences that could materially and adversely affect our operating results and financial condition; we contract with a single manufacturer for the purchase of microarray chips for certain tests, and the failure of this manufacturer to supply sufficient quantities on a timely basis could have a material adverse effect on our business; and other risk factors discussed under the heading "Risk Factors" in Rosetta's most recently filed Annual Report on Form 20-F, as filed with the SEC. In addition, any forward-looking statements represent Rosetta's views only as of the date of this release and should not be relied upon as representing its views as of any subsequent date. Rosetta does not assume any obligation to update any forward-looking statements unless required by law.


PHILADELPHIA, PA and REHOVOT, ISRAEL / ACCESSWIRE / May 5, 2017 / Rosetta Genomics Ltd. (NASDAQ: ROSG), a genomic diagnostics company that improves treatment decisions by providing timely and accurate diagnostic information to physicians, announces that the Company has signed an exclusive distribution agreement with Cytolog Laboratories (Cytolog) for the sales and marketing of RosettaGX RevealTM (Reveal), a first-of-its-kind microRNA assay for the classification of indeterminate thyroid nodules, in Brazil. Cytolog is a Brazilian-based diagnostics laboratory that specializes in fine needle aspiration examinations. Cytolog already has the regulatory approvals to send biological samples outside of Brazil and expects to begin utilizing RosettaGX Reveal effective immediately. "We are delighted to partner with Rosetta Genomics to bring RosettaGX Reveal to patients in Brazil who have an indeterminate thyroid cancer diagnosis. In addition to its excellent performance, Reveal has a significant advantage to current assays on the market because it can work off the same cytology slides that were created to perform the initial diagnosis, thus eliminating the risks, added patient stress, and unnecessary pain associated with additional fine needle passes," stated Fabiano Callegari, M.D., pathologist and principal of Cytolog. "We look forward to bringing Reveal to the Brazilian market as many patients with indeterminate thyroid cancer undergo surgery as a precaution despite the fact that up to 80% of these cases are benign. This exposes patients to unnecessary surgical risk and costs the healthcare system millions of dollars," added Dr. Callegari. According to Surveillance, Epidemiology & End Results (SEER), a U.S. National Cancer Institute program that collects incidence and survival data from cancer registries, the incidence of thyroid cancer has tripled in the past 35 years. In Brazil, the National Cancer Institute (INCA) estimated 9,200 new cases in 2014, 8,050 of them in women. This is largely the result of improved diagnosis due to the increased use of ultrasound scanning. "We are particularly pleased to expand access for Reveal to the Brazilian market where the incidence of thyroid cancer diagnosis is growing," stated Kenneth A. Berlin, President and Chief Executive Officer of Rosetta Genomics. "Unlike other assays in the space, Reveal does not require fresh tissue and therefore does not require special logistics for sample handling and shipment. These simple logistics make it ideally suited for international distribution agreements like this one. We look forward to working with Cytolog, a premier laboratory with a reputation as a leader in the field of fine needle aspirates. This is our first distribution agreement in South America and it marks a significant milestone in our strategic goal to expand global access for Reveal." Cytolog is a Brazilian-based laboratory that specializes in fine needle aspiration examinations, which today is a reference in precise diagnoses, equaled to the best medical centers in the world and certified by the Brazilian Societies of Pathology and Cytopathology. The evolution of medicine has been generous to patients, not only for seeking healing for their illnesses but also for providing an early diagnosis. Proof of this is the fine needle aspiration examination, which can replace the surgical biopsy, making the procedure quick, simple and well accepted by patients. This examination consists of the cytological analysis of thyroid nodules, salivary glands, breast, and lymph nodes, among other lesions. Cytolog has very strong human values and therefore offers personalized accompaniment to all its patients, providing much more comfort and well-being. Rosetta is pioneering the field of molecular diagnostics by offering rapid and accurate diagnostic information that enables physicians to make more timely and informed treatment decisions to improve patient care. Rosetta has developed a portfolio of unique diagnostic solutions for oncologists, urologists, endocrinologists, cytopathologists and other specialists to help them deliver better care to their patients. RosettaGX Reveal™, a Thyroid microRNA Classifier for the diagnosis of cancer in thyroid nodules, as well as the full RosettaGX™ portfolio of cancer testing services are commercially available through the Company's Philadelphia, PA- and Lake Forest, CA-based CAP-accredited, CLIA-certified labs. Various statements in this release concerning Rosetta's future expectations, plans and prospects including, but not limited to bringing Reveal to the Brazilian market and expanding global access and statements containing the words "expect," "believe," "will," "may," "should," "project," "estimate," "anticipated," "scheduled," and like expressions, and the negative thereof, constitute forward-looking statements for the purposes of the safe harbor provisions under The Private Securities Litigation Reform Act of 1995. Actual results may differ materially from those indicated by these forward-looking statements as a result of various risks and uncertainties, including, but are not limited to the following: we will require substantial additional funds to continue our operations and, if additional funds are not available, we may need to significantly scale back or cease our operations; we have a history of losses and may never be profitable; if we are unable to expand sales of our diagnostic tests in the United States, it would have a material adverse effect on our business and financial condition; the intensely competitive biotechnology market could diminish demand for our tests and products; the market may not be receptive to any diagnostic tests or therapeutic products using our microRNA technology; we currently have limited sales, marketing or distribution experience and may in the future depend significantly on third parties to commercialize microRNA-based diagnostic tests or therapeutic products we may develop; we are largely dependent upon our distributors for the success of commercialization of our current diagnostic tests; health insurers and other third-party payors may decide not to cover our diagnostic products or may provide inadequate reimbursement, which could jeopardize our commercial prospects; because of Medicare billing rules, we may not receive reimbursement for all tests provided to Medicare patients; if we fail to comply with our obligations under any licenses or related agreements, we could lose license rights that may be necessary for developing microRNA-based diagnostics and therapeutics; if we fail to comply with the complex federal, state, local and foreign laws and regulations that apply to our business, we could suffer severe consequences that could materially and adversely affect our operating results and financial condition; we contract with a single manufacturer for the purchase of microarray chips for certain tests, and the failure of this manufacturer to supply sufficient quantities on a timely basis could have a material adverse effect on our business; and other risk factors discussed under the heading "Risk Factors" in Rosetta's most recently filed Annual Report on Form 20-F, as filed with the SEC. In addition, any forward-looking statements represent Rosetta's views only as of the date of this release and should not be relied upon as representing its views as of any subsequent date. Rosetta does not assume any obligation to update any forward-looking statements unless required by law. PHILADELPHIA, PA and REHOVOT, ISRAEL / ACCESSWIRE / May 5, 2017 / Rosetta Genomics Ltd. (NASDAQ: ROSG), a genomic diagnostics company that improves treatment decisions by providing timely and accurate diagnostic information to physicians, announces that the Company has signed an exclusive distribution agreement with Cytolog Laboratories (Cytolog) for the sales and marketing of RosettaGX RevealTM (Reveal), a first-of-its-kind microRNA assay for the classification of indeterminate thyroid nodules, in Brazil. Cytolog is a Brazilian-based diagnostics laboratory that specializes in fine needle aspiration examinations. Cytolog already has the regulatory approvals to send biological samples outside of Brazil and expects to begin utilizing RosettaGX Reveal effective immediately. "We are delighted to partner with Rosetta Genomics to bring RosettaGX Reveal to patients in Brazil who have an indeterminate thyroid cancer diagnosis. In addition to its excellent performance, Reveal has a significant advantage to current assays on the market because it can work off the same cytology slides that were created to perform the initial diagnosis, thus eliminating the risks, added patient stress, and unnecessary pain associated with additional fine needle passes," stated Fabiano Callegari, M.D., pathologist and principal of Cytolog. "We look forward to bringing Reveal to the Brazilian market as many patients with indeterminate thyroid cancer undergo surgery as a precaution despite the fact that up to 80% of these cases are benign. This exposes patients to unnecessary surgical risk and costs the healthcare system millions of dollars," added Dr. Callegari. According to Surveillance, Epidemiology & End Results (SEER), a U.S. National Cancer Institute program that collects incidence and survival data from cancer registries, the incidence of thyroid cancer has tripled in the past 35 years. In Brazil, the National Cancer Institute (INCA) estimated 9,200 new cases in 2014, 8,050 of them in women. This is largely the result of improved diagnosis due to the increased use of ultrasound scanning. "We are particularly pleased to expand access for Reveal to the Brazilian market where the incidence of thyroid cancer diagnosis is growing," stated Kenneth A. Berlin, President and Chief Executive Officer of Rosetta Genomics. "Unlike other assays in the space, Reveal does not require fresh tissue and therefore does not require special logistics for sample handling and shipment. These simple logistics make it ideally suited for international distribution agreements like this one. We look forward to working with Cytolog, a premier laboratory with a reputation as a leader in the field of fine needle aspirates. This is our first distribution agreement in South America and it marks a significant milestone in our strategic goal to expand global access for Reveal." Cytolog is a Brazilian-based laboratory that specializes in fine needle aspiration examinations, which today is a reference in precise diagnoses, equaled to the best medical centers in the world and certified by the Brazilian Societies of Pathology and Cytopathology. The evolution of medicine has been generous to patients, not only for seeking healing for their illnesses but also for providing an early diagnosis. Proof of this is the fine needle aspiration examination, which can replace the surgical biopsy, making the procedure quick, simple and well accepted by patients. This examination consists of the cytological analysis of thyroid nodules, salivary glands, breast, and lymph nodes, among other lesions. Cytolog has very strong human values and therefore offers personalized accompaniment to all its patients, providing much more comfort and well-being. Rosetta is pioneering the field of molecular diagnostics by offering rapid and accurate diagnostic information that enables physicians to make more timely and informed treatment decisions to improve patient care. Rosetta has developed a portfolio of unique diagnostic solutions for oncologists, urologists, endocrinologists, cytopathologists and other specialists to help them deliver better care to their patients. RosettaGX Reveal™, a Thyroid microRNA Classifier for the diagnosis of cancer in thyroid nodules, as well as the full RosettaGX™ portfolio of cancer testing services are commercially available through the Company's Philadelphia, PA- and Lake Forest, CA-based CAP-accredited, CLIA-certified labs. Various statements in this release concerning Rosetta's future expectations, plans and prospects including, but not limited to bringing Reveal to the Brazilian market and expanding global access and statements containing the words "expect," "believe," "will," "may," "should," "project," "estimate," "anticipated," "scheduled," and like expressions, and the negative thereof, constitute forward-looking statements for the purposes of the safe harbor provisions under The Private Securities Litigation Reform Act of 1995. Actual results may differ materially from those indicated by these forward-looking statements as a result of various risks and uncertainties, including, but are not limited to the following: we will require substantial additional funds to continue our operations and, if additional funds are not available, we may need to significantly scale back or cease our operations; we have a history of losses and may never be profitable; if we are unable to expand sales of our diagnostic tests in the United States, it would have a material adverse effect on our business and financial condition; the intensely competitive biotechnology market could diminish demand for our tests and products; the market may not be receptive to any diagnostic tests or therapeutic products using our microRNA technology; we currently have limited sales, marketing or distribution experience and may in the future depend significantly on third parties to commercialize microRNA-based diagnostic tests or therapeutic products we may develop; we are largely dependent upon our distributors for the success of commercialization of our current diagnostic tests; health insurers and other third-party payors may decide not to cover our diagnostic products or may provide inadequate reimbursement, which could jeopardize our commercial prospects; because of Medicare billing rules, we may not receive reimbursement for all tests provided to Medicare patients; if we fail to comply with our obligations under any licenses or related agreements, we could lose license rights that may be necessary for developing microRNA-based diagnostics and therapeutics; if we fail to comply with the complex federal, state, local and foreign laws and regulations that apply to our business, we could suffer severe consequences that could materially and adversely affect our operating results and financial condition; we contract with a single manufacturer for the purchase of microarray chips for certain tests, and the failure of this manufacturer to supply sufficient quantities on a timely basis could have a material adverse effect on our business; and other risk factors discussed under the heading "Risk Factors" in Rosetta's most recently filed Annual Report on Form 20-F, as filed with the SEC. In addition, any forward-looking statements represent Rosetta's views only as of the date of this release and should not be relied upon as representing its views as of any subsequent date. Rosetta does not assume any obligation to update any forward-looking statements unless required by law.


Prague, Czech Republic - 26 May 2017: Researchers in the UK and Peru have developed a faster and cheaper cardiac imaging test that can be used in developing countries, according to the results of the INCA-Peru study presented today at EuroCMR 2017.1 The scan is three times faster, less than one-fifth of the cost, and changed clinical management in 33% of patients. Cardiovascular magnetic resonance (CMR) is the gold standard for diagnosing many cardiovascular diseases. It is used to measure the structure and function of the heart, and to assess scarring on the heart muscle after a heart attack to predict if another will occur. Lead author Dr Katia Menacho, a cardiologist from Peru and PhD student at University College London (UCL), UK, said: "In Peru just two public hospitals offer CMR - each performs 12 scans on one day a week. Five private hospitals provide CMR at $USD 600-800 per scan. Public hospitals without CMR refer to the private sector and it takes up to three months to approve the paperwork, delaying diagnosis and treatment." The Impact of Non-invasive CMR Assessment (INCA)-Peru study was set up to develop and test an ultrafast CMR protocol for wider uptake in Peru. The study was an international academic and political collaboration between Peru, the UK, the USA and Brazil. An initial protocol was developed at UCL which reduced average scan time from 60 to ten minutes. It was successfully used in Thailand to assess cardiac and liver iron overload in patients with thalassaemia major.2 The protocol was adapted for the INCA-Peru study by adding the contrast agent gadolinium which is critical for the detection of cardiac fibrosis and infarction, and the diagnosis of various cardiovascular diseases. It was tested in a pilot study of 50 patients at UCL and each scan took 15 to 20 minutes. For the current analysis, the ultrafast CMR protocol using contrast was tested over two days at two hospitals in Peru, accompanied by training for local cardiologists, radiologists, and technicians. The study included 100 patients with suspected cardiomyopathy and 11 healthy controls. All of the patients were under local cardiology care and had never been assessed by CMR. There were no scan-related complications. All scans were diagnostic, although two patients had a repeat scan (one due to failure of contrast delivery and one to confirm unexpected cardiac amyloidosis). Gadolinium based contrast agents were administered to 95% of patients (5% of patients had iron overload). Each ultrafast CMR scan took 18 minutes on average and cost $USD 150. The most common underlying diagnoses were hypertrophic cardiomyopathy (21%), non-ischaemic dilated cardiomyopathy (17%), and ischaemic cardiomyopathy (11%), plus 20 other diagnoses including tumours, congenital heart disease, myocardial iron overload, amyloidosis, vasculitis, and apical thrombus. CMR had an impact on the clinical management of 33% of patients. CMR revealed a new, unsuspected diagnosis in 20% of patients, leading to a complete change in management. In 30% of patients no further cardiac testing was needed (coronary angiography, computed tomography, cardiac biopsy). "We showed that this ultrafast CMR protocol can be used to accurately diagnosis patients leading to changes in clinical management," said Dr Menacho. "The protocol takes one-third of the standard scan time at a fraction of the cost." INCA-Peru researchers will follow-up the patients for two years to evaluate the impact of diagnosis by ultrafast CMR on long-term morbidity and mortality. Dr Menacho said: "Accurate diagnoses provided by CMR have reduced morbidity and mortality in Europe and we hope to find the same in Peru. If we show long-term benefits on prognosis we will seek support from the Peruvian government to provide ultrafast CMR at more hospitals in Peru. The accurate diagnosis provided by CMR should lead to more targeted patient care and better outcomes."


MitoSOX Red mitochondrial superoxide indicator was from Life Technologies. MitoTEMPO, N-acetylcysteine (NAC), Nocodazole, DHEA and DCA were from Sigma. The IDH1 inhibitor 321 and control compound 990 were from GlaxoSmithKline12. Cell lines were identified using DNA fingerprinting and confirmed to be mycoplasma free. H460, A549, HBEC30 and HCC4017 cells were provided by J. D. Minna, UT Southwestern. MCF7 cells were provided by David A. Boothman, UT Southwestern. HT-29 cells were provided by Jared Rutter, University of Utah. H460 and A549 cells were cultured in RPMI supplemented with penicillin/streptomycin, 5% fetal bovine serum (FBS), l-glutamine (4 mM) and 1 mM HEPES. HT-29 and MCF7 were cultured in DMEM supplemented with penicillin/streptomycin, 10% fetal bovine serum (FBS), l-glutamine (4 mM) and 1 mM HEPES. HBEC30 and HCC4017 cells were cultured in defined medium5. Dishes with an Ultra-Low Attachment surface were used for suspension and spheroid culture. Identical culture medium was used for monolayer and spheroid culture. For spheroids, 2 × 105 H460 cells were plated in a 10 cm Ultra-Low Attachment dish. The medium was changed on days 4, 6 and 7 of culture, by centrifuging at 50g for 3 min, then gently resuspending in fresh medium. For labelling assays, the spheroids were resuspended in medium containing 13C-labelled nutrients, and the time of resuspension was considered time 0 of the labelling period. IDH1, IDH2, IDH3 and SLC25A1-deficient H460 cell lines were generated using the CRISPR/Cas9 system21. Wild type clones were selected from both the control vector (Vector) and targeting vector transfections (WT). In order to control for variations among individual clones, 4 to 5 clones were pooled together, and different pools for each targeted gene were used for further experiments. Cell proliferation was measured by counting cells after trypsinization. DNA content was also used to monitor monolayer cell growth, as previously described22. Spheroid size was determined by measuring the maximum cross-sectional area of individual spheroids using ImageJ software. Glucose, lactate, glutamine and glutamate were measured in culture medium using an automated electrochemical analyser (BioProfile Basic-4 analyser, NOVA). Ammonia was measured using an enzymatic assay (Megazyme). Nutrients labelled with 13C, 2H or 15N were purchased from Cambridge Isotope Laboratories. Stable isotope tracing experiments to determine isotopologue distributions in soluble metabolites and fatty acids were performed as described previously22, 23. For deuterium tracing, H460 clones were engineered to express the IDH2-R172K mutant (mtIDH2) under control of doxycycline16. Clones lacking wild-type IDH1 or IDH2, or containing both, were generated. Spheroids were cultivated for 7 days, then doxycycline (0.2 μg ml−1) was added for 24 h to induce mtIDH2. On day 8, spheroids were cultured in RPMI containing 10 mM [3-2H]glucose and unlabelled glutamine for 6 h. Mitochondria were prepared with the Qproteome Mitochondria Isolation Kit (Qiagen). Isotope tracing was modified from a previously described procedure22. Mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM P , pH 7.4) supplied with indicated nutrients and tracer. For glutamine tracing, 40 μM [U-13C]glutamine and 40 μM unlabelled pyruvate were added to the assay buffer. For citrate tracing, 40 μM [U-13C]citrate with or without 40μM unlabelled glutamine and 40μM unlabelled pyruvate were added to the assay buffer. Mitochondria were incubated in the tracing buffer for 10 min, at 30 °C with 500 r.p.m. agitation in a heat block. Steady state metabolic fluxes were calculated by combining extracellular flux rates (glucose/glutamine utilization, lactate/alanine/glutamate secretion) and 13C mass isotopologue distributions (MIDs) for citrate, glutamate, fumarate, malate, aspartate and palmitate, using the INCA software package24, which applies an elementary metabolite unit framework to efficiently simulate MIDs25, 26. We developed reaction networks describing the stoichiometry and carbon transitions of central carbon metabolism (Supplementary Table 2), with assumptions as previously described4 and summarized below. Parallel labelling data from cultures fed [1-13C]glutamine, [5-13C]glutamine, [U-13C]glutamine or [U-13C]glucose were used to simultaneously fit the same network model to estimate intracellular fluxes. Data used in metabolic flux analysis for monolayer and spheroid cultures are reported in Supplementary Table 3. To ensure that a global minimum of fluxes was identified, flux estimations were initiated from random values and repeated a minimum of 50 times. A chi-square test was applied to test goodness-of-fit, and accurate 95% confidence intervals were calculated by assessing the sensitivity of the sum of squared residuals to flux parameter variations. Extended Data Table 1 contains the degrees of freedom and sum-of-squared residuals (SSR) for the best fit model and the lower and upper bounds of 95% confidence intervals for all fluxes. Whole cells/spheroids or mitochondrial lysates were prepared in RIPA buffer and quantified using the BCA Protein Assay (Thermo Scientific). Proteins were separated on 4–20% SDS–PAGE gels, transferred to PVDF membranes, and probed with antibodies against IDH1 (ab94571), IDH2 (ab55271), IDH3 (ab58641) from Abcam, PDHα (#459400, Thermo), PDHα-pSer293 (AP1062), GAPDH (AB2302) from Millipore, PDK1 (#3820), Hif1α (#3716) from Cell Signaling, CTP (sc-86392), AIF (sc-13116) from Santa Cruz Biotechnology and Actin (A3853, Sigma). Day 7 H460 spheroids were cultured under normoxia (21% oxygen) or hypoxia (1% oxygen) for 16 h. The spheroids were then treated with 100 μM EF5 compound for 3 h, fixed in 4% paraformaldehyde, and embedded in OCT for frozen sectioning. 10 μm spheroid sections were stained with EF5 antibody as previously described27. Cytosolic and mitochondrial ROS levels were measured with the organelle-specific HyPer system as previously described15, 28. Briefly, trypsinized H460 cells were transfected with HyPer-cyto or HyPer-mito vectors. Half of the transfected cells were allowed to attach as a monolayer, and the other half were cultured in suspension using Ultra-Low Attachment dishes. Images were acquired 48 h after transfection, and ROS levels were calculated as the ratio of fluorescence at 488 nm and 405 nm. For monolayer respiration assays, H460 cells were plated in growth media at 3 × 104 cells per well in XF24 microplates (Seahorse Bioscience; Billerica, MA) 24 h before the assay following manufacturer’s recommendations for cell seeding. Growth media was changed to XF assay media and the plates were incubated at 37 °C in a non-CO incubator for 45 min before starting the assay. To normalize the data, the cells were trypsinized and counted by haemocytometer or ViCELL automated cell counter (Beckman Coulter). To measure respiration in spheroids, H460 Spheroids were grown for 3 days in 96-well Ultra-Low Attachment round bottom plates (Sigma; Cat# CLS7007) starting with 2 × 103 cells per well, resulting in an average spheroid diameter of 401 ± 13 μm. For the respiration assay, spheroids were transferred to a Poly-d-lysine-treated XFe96 Spheroid Microplate (Seahorse Bioscience cat# 102959-100) containing 37 °C XF assay media at pH 7.4. Following each assay, the spheroid diameter was measured using images acquired on a Cytation 3 Cell Imaging Reader (BioTek; Winooski, VT). Volume was then calculated using the equation v = 4/ πr3. To normalize the data, the cell number of each spheroid was calculated by dividing the spheroid volume by the single-cell volume (average diameter of H460 cells = 14 μm). This method of calculating spheroid cell number was independently validated in parallel cultures by digesting spheroids with trypsin and manually counting the cells with a haemocytometer. IDH1 was generated as previously described29. IDH2 was expressed in Sf 9 cells by baculovirus infection. Cells were lysed in lysis buffer (50 mM Tris, pH 7.5, 300 mM NaCl, 10% glycerol, 1% Triton) by Avestin emulsiflex C50. The supernatants were mixed with anti-Flag resin at 4 °C overnight and then eluted with addition of 100 μg ml−1 Flag peptide in lysis buffer (without Triton). The eluate was concentrated and loaded onto a Superdex 200 size exclusion column and eluted with sizing buffer (25 mM Tris, 100 mM NaCl, 1 mM DTT, pH 7.5). Fractions containing dimer were pooled and concentrated for use in kinetics. IDH activity was assessed by measuring the evolution of NADPH abundance using a coupled diaphorase/resazurin fluorescence assay30. Reactions were conducted at room temperature in 384-well Greiner black microtitre plates in a total volume of 10 μl of assay buffer. Final compound concentrations were typically varied from 5 to 100,000 nM; the isocitrate concentration was fixed at 10 μM, the NADP+ concentration was fixed at 5 μM, and IDH was fixed at 0.1 nM. Reactions were conducted in quadruplicate and run kinetically. After each addition, plates were centrifuged for 60 s to ensure complete mixing of reagents. Data were fit to the following equation to determine the IC : where y is the % of normalized enzyme activity, x is the concentration of inhibitor, and s is the Hill slope factor. No statistical methods were used to predetermine sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment. Experiments in Figs 2a, 2b, 4f, and Extended Data Figs 5d, 7d and 8e were performed twice, and all other experiments were performed 3 times or more. Variation is always indicated using standard deviation. To assess the significance of differences among cultures and conditions, a two-tailed Welch’s unequal variances t-test was used to assess the significance between two groups. For three or more groups, a one-way ANOVA followed by Dunnett’s multiple comparisons test was performed. Before applying ANOVA, we first tested whether there was homogeneity of variation among the groups (as required for ANOVA) using the Brown–Forsythe test. Where the assumption of equal variance was violated, a log transformation was applied to the data before analysis. In a few cases, when significant differences in variation among groups persisted after transformation, we used Welch’s unequal variances t-test followed by multiple-comparison correction.


Antunes H.S.,Instituto Nacional Of Cancer Inca | Herchenhorn D.,INCA | Small I.A.,Instituto Nacional Of Cancer Inca | Araujo C.M.M.,INCA | And 7 more authors.
Radiotherapy and Oncology | Year: 2013

Background Oral mucositis (OM) is a complication of chemoradiotherapy treatment of head and neck squamous cell carcinoma (HNSCC) patients with no effective therapy. This study was designed to assess the efficacy of preventive low-level laser therapy (LLLT) in reducing the incidence of grade 3-4 OM. Material and methods From June 2007 to December 2010, 94 HNSCC patients entered a prospective, randomized, double-blind, placebo-controlled phase III trial. Chemoradiotherapy consisted of conventional radiotherapy plus concurrent cisplatin every 3 weeks. A diode InGaAlP (660 nm-100 mW-1 J-4 J/cm2) was used. OM evaluation was performed by WHO and OMAS scales and quality of life by EORTC questionnaires (QLQ). Results A six-fold decrease in the incidence of grades 3-4 OM was detected in the LLLT group compared to the placebo; (6.4% versus 40.5%). LLLT impacted the incidence of grades 3-4 OM to a relative risk ratio of 0.158 (CI 95% 0.050-0.498). After treatment QLQ-C30 showed, differences favoring LLLT in physical, emotional functioning, fatigue, and pain; while the QLQ-H&N35 showed improvements in LLLT arm for pain, swallowing, and trouble with social eating. Conclusion Preventive LLLT in HNSCC patients receiving chemoradiotherapy is an effective tool for reducing the incidence of grade 3-4 OM. Efficacy data were corroborated by improvements seen in quality of life. © 2013 Elsevier Ireland Ltd. All rights reserved.


News Article | October 28, 2016
Site: www.sciencedaily.com

Microplastics are increasingly seen as an environmental problem of global proportions. While the focus to date has been on microplastics in the ocean and their effects on marine life, microplastics in soils have largely been overlooked. Researchers are concerned about the lack of knowledge regarding potential consequences of microplastics in agricultural landscapes from application of sewage sludge. Sewage sludge is in principle waste, but it can also represent a resource in agriculture and horticulture. Fertilizer based on sludge contains valuable nutrients, but sustainable use requires that the levels of undesirable substances in the sludge is kept under control. Waste water treatment plants receive large amounts of microplastics emitted from households, industry and surface run-off in urban areas. Most of these microplastics accumulate in the sewage sludge. Today, sludge from municipal sewage treatment plants is applied to agricultural areas as a supplement to traditional fertilizers. These applications are generally well regulated as sludge might contain hazardous substances of different sorts. Microplastics are however not currently on the regulatory agenda for the use of sludge in agriculture. The potential consequences for sustainability and food security have not been adequately analyzed. These concerns have been expressed in an article recently published in the journal Environmental Science & Technology. The researchers behind the article are Luca Nizzetto and Sindre Langaas from the Norwegian Institute for Water Research (NIVA) and Martyn Futter from the Swedish University of Agricultural Sciences (SLU) in Uppsala. "We have found figures from the Nordic countries suggesting that a large fraction of all the microplastics generated in Western societies tend to end up in the sludge in wastewater treatment plants," says Nizzetto. Via the sludge the particles are transferred to agricultural soils. The amount of sewage sludge used as fertilizer varies greatly from country to country. In Europe and North America approximately 50 % of this sludge is reused as fertilizer on average. According to Statistics Norway, about two thirds of the sludge is reused in this manner. Nizzetto et al estimates that between 110,000 and 730,000 tons of microplastics are transferred every year to agricultural soils in Europe and North America, comprehensively. This is more than the estimated total burden of microplastics currently present in ocean water. These figures are of concern since the effects of microplastics accumulating in agricultural soils are unknown. "We have very little knowledge on the effect of microplastics on soil organisms, and their impact on farm productivity and food safety is unknown." The first simulation of microplastic fate on land and rivers In an earlier study from the same authors, and researchers of Oxford University, the first mathematical model describing the dynamics of microplastics' fate in terrestrial environments and rivers was presented. Due to a lack of empirical data on microplastics emissions and concentrations in soils and the stream system, this study was conceived to provide a purely theoretical, nevertheless rigorous, assessment of microplastics circulation. The model is called INCA Microplastics, and simulations have showed a strong influence of meteorological conditions and river characteristics and flows in controlling the export of microplastics from agricultural soils and their transport to the ocean. Application of sewage sludge to soils likely represent a considerable source of microplastics to the coastal and ocean environments. Similar predictions for the transport of microplastics in rivers were independently confirmed by a follow-up study by Besseling et al. INCA Microplastics is an important tool for risk assessment and evaluating sludge management scenarios. It is the first model able to simulate microplastic applications to land, and the consequent fate of these materials in soils and surface waters. The consequences of transfers of microplastics from urban waste water to agricultural soil barely have been considered by researchers and authorities, particularly in lieu of the extended attention directed at microplastics in the ocean. "Clearly further research is needed to get an overview of the problem -- and to find solutions -- so that the growing need in the community for recycling and so-called circular economy can be safeguarded," Luca Nizzetto says.


News Article | December 21, 2016
Site: www.prnewswire.com

VANCOUVER, Dec. 21, 2016 /PRNewswire/ - INCA ONE GOLD CORP. (TSX.V: IO) ("Inca One" or the "Company") is pleased to provide the following update on the Company's operations from its wholly owned subsidiary Chala One SAC ("Chala One"). The Company's 90 day ramp-up has now passed its...


News Article | November 21, 2016
Site: www.prnewswire.com

VANCOUVER, Nov. 21, 2016 /PRNewswire/ - INCA ONE GOLD CORP. (TSX.V: IO) ("Inca One" or the "Company") is pleased to provide the following ramp-up update on operations from its wholly owned subsidiary Chala One SAC ("Chala One"). In the initial 31 day period from the commencement of...


LONDON, UK, February 28, 2017 – Superfast rural connectivity schemes using fixed wireless broadband technology will be pivotal for alternative network providers (Altnets) hoping to win a major slice of the latest BDUK funding programme, operators were told today. Speaking as part of the Independent Network Co-operative Association (INCA) seminar “Bidding For BDUK Projects & The Better Broadband Scheme,” Dan McCarthy, Cambium Networks’ UK sales manager for the UK, Ireland and Nordics, highlighted the qualities of wireless connectivity as being particularly suitable for the UK market. Cambium Networks is a leading provider of wireless networking solutions, deployed by network operators, public and private, of all sizes around the world. “As the Government looks to meet superfast connectivity targets, fixed wireless technology will play a key role in building the networks to enable ubiquitous broadband across the country,” said McCarthy. “Altnets which utilise this technology will find themselves in a very good position to secure funding due to the many advantages wireless networks offer to operators; they are quick, simple and cost-effective to deploy and enable the high-capacity connections stipulated for Superfast coverage of 30 Mbps downlink and 6 Mbps uplink.” Despite the suitability of Fixed Wireless Access, McCarthy went on to warn operators that not all solutions are the same when it comes to being successful with BDUK bids. “When it comes to wireless solutions, it is easy to see them all as being equal but some are more equal than others,” said McCarthy. “When choosing a vendor, operators who select one offering BDUK-compliant equipment will find themselves at an advantage when applying for funding. A simple and fast migration path from Superfast broadband to ultrafast broadband, such as the one offered by our portfolio, will also stand them in good stead.” McCarthy demonstrated the sort of success such a solution can bring with a Cambium Networks customer – Quickline, which delivers fast reliable Internet connectivity to Business, Public Sector and Residential customers via an independent Fibre and Fixed Wireless Access network, alongside its partner networks. Cambium Networks provided its PMP 450 and ePMP™ Elevate solutions to boost Quickline’s networks in Yorkshire, enabling 50Mbps throughput to more than 1,000 subscribers to be connected to superfast broadband. Quickline’s Managing Director Steve Jagger said: “The expertise and experience which the Cambium Networks’ team brought to this project really helped its success. We were also very impressed by the product set, which has the advantage of simplifying deployment and has predictable, reliable NGA characteristics required for State Aid Compliance.” McCarthy also highlighted how a number of grants have recently been awarded to Altnets under the BDUK scheme. Currently, more than £100m of funding is available to suppliers able to serve rural areas with superfast broadband services, with £400m of gainshare funding from the original BDUK contracts potentially available. INCA is working with BDUK to help more Altnets take advantage of the funding, with today’s seminar part of these efforts. The second part of the event – NGA Compliance for Local Access Providers – will take place tomorrow at the De Vere Grand Connaught Rooms in London. About Cambium Networks: Cambium Networks is a leading global provider of trusted wireless solutions that connect the unconnected – People, Places and Things. Through its extensive portfolio of reliable, scalable and secure wireless narrowband and broadband platforms, Cambium Networks makes it possible for all service providers and industrial, enterprise and government network operators to build affordable, reliable, high-performance connectivity. The company currently has over six million radios deployed in thousands of demanding networks in more than 150 countries. Headquartered outside Chicago and with R&D centers in the U.S., U.K. and India, Cambium Networks sells through a range of trusted global distributors. For more information, visit www.cambiumnetworks.com and www.connectingtheunconnected.org. For more information: Dan McCarthy Sales Manager UK, Ireland and Nordics E-mail: dan.mccarthy@cambiumnetworks.com


News Article | February 24, 2017
Site: marketersmedia.com

— The Global Maca Extract Market Research Report 2017 is a professional and in-depth study on the current state of the Maca Extract industry. In a word, This report studies Maca Extract in Global market, especially in North America, Europe, China, Japan, Southeast Asia and India, focuses on top manufacturers in global market, with capacity, production, price, revenue and market share for each manufacturer. Key companies included in this research are Koken, Peruvian Nature, ZANACEUTICA, INCA HEALTH, Pebani, StandPeru, Phyto Life Sciences, Jiaherb, Pioneer Herbs, Green Life, Yuansn Biological, Bettering, Yongyuan Bio-Tech, Naturalin, Berbchem Biotech, Tengmai and Huike. Market Segment by Region, this report splits Global into several key Region, with sales, revenue, market share and growth rate of Maca Extract in these regions, from 2011 to 2022 (forecast), like North America, Europe, China, Japan, Southeast Asia and India. Firstly, Maca Extract Market On the basis of product, this report displays the production, revenue, price, market share and growth rate of each type, primarily split into White to Yellow, Light Pink to Dark Purple and Light Gray to Dark Gray. On the basis on the end users/applications, this report focuses on the status and outlook for major applications/end users, consumption (sales) , market share and growth rate of Maca Extract for each application, including Health Drugs, Healthy Foods and Nutritional Supplements. 7.1 Koken 7.1.1 Company Basic Information, Manufacturing Base, Sales Area and Its Competitors 7.1.2 Maca Extract Product Category, Application and Specification 7.1.2.1 Product A 7.1.2.2 Product B 7.1.3 Koken Maca Extract Capacity, Production, Revenue, Price and Gross Margin (2012-2017) 7.1.4 Main Business/Business Overview 7.2 Peruvian Nature 7.2.1 Company Basic Information, Manufacturing Base, Sales Area and Its Competitors 7.2.2 Maca Extract Product Category, Application and Specification 7.2.2.1 Product A 7.2.2.2 Product B 7.2.3 Peruvian Nature Maca Extract Capacity, Production, Revenue, Price and Gross Margin (2012-2017) 7.2.4 Main Business/Business Overview 7.3 ZANACEUTICA 7.3.1 Company Basic Information, Manufacturing Base, Sales Area and Its Competitors 7.3.2 Maca Extract Product Category, Application and Specification 7.3.2.1 Product A 7.3.2.2 Product B 7.3.3 ZANACEUTICA Maca Extract Capacity, Production, Revenue, Price and Gross Margin (2012-2017) 7.3.4 Main Business/Business Overview Table Production Base and Market Concentration Rate of Raw Material Figure Price Trend of Key Raw Materials Table Key Suppliers of Raw Materials Figure Manufacturing Cost Structure of Maca Extract Figure Manufacturing Process Analysis of Maca Extract Figure Maca Extract Industrial Chain Analysis Table Raw Materials Sources of Maca Extract Major Manufacturers in 2016 Table Major Buyers of Maca Extract Table Distributors/Traders List Figure Global Maca Extract Capacity, Production and Growth Rate Forecast (2017-2022) Figure Global Maca Extract Revenue and Growth Rate Forecast (2017-2022) Figure Global Maca Extract Price and Trend Forecast (2017-2022) Table Global Maca Extract Production Forecast by Region (2017-2022) Figure Global Maca Extract Production Market Share Forecast by Region (2017-2022) Table Global Maca Extract Consumption Forecast by Region (2017-2022) Figure Global Maca Extract Consumption Market Share Forecast by Region (2017-2022) Figure North America Maca Extract Production and Growth Rate Forecast (2017-2022) Figure North America Maca Extract Revenue and Growth Rate Forecast (2017-2022) Table North America Maca Extract Production, Consumption, Export and Import Forecast (2017-2022) Figure Europe Maca Extract Production and Growth Rate Forecast (2017-2022) Figure Europe Maca Extract Revenue and Growth Rate Forecast (2017-2022) Table Europe Maca Extract Production, Consumption, Export and Import Forecast (2017-2022) Figure China Maca Extract Production and Growth Rate Forecast (2017-2022) Figure China Maca Extract Revenue and Growth Rate Forecast (2017-2022) Table China Maca Extract Production, Consumption, Export and Import Forecast (2017-2022) Figure Japan Maca Extract Production and Growth Rate Forecast (2017-2022) Figure Japan Maca Extract Revenue and Growth Rate Forecast (2017-2022) Table Japan Maca Extract Production, Consumption, Export and Import Forecast (2017-2022) Figure Southeast Asia Maca Extract Production and Growth Rate Forecast (2017-2022) Figure Southeast Asia Maca Extract Revenue and Growth Rate Forecast (2017-2022) Table Southeast Asia Maca Extract Production, Consumption, Export and Import Forecast (2017-2022) Figure India Maca Extract Production and Growth Rate Forecast (2017-2022) Figure India Maca Extract Revenue and Growth Rate Forecast (2017-2022) Table India Maca Extract Production, Consumption, Export and Import Forecast (2017-2022) For more information, please visit http://www.reportsweb.com/global-maca-extract-market-research-report-2017

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