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Objectives: Currently, no systemic treatments are approved for patients with recurrent and/or metastatic (R/M) adenoid cystic carcinoma (ACC). PRT543, a protein arginine methyltransferase 5 inhibitor that downregulates NOTCH1 and MYB signalling in tumours, is a potential candidate for R/M ACC treatment. We report the safety, tolerability and preliminary efficacy of PRT543 in a dose-expansion cohort of patients with R/M ACC., Materials and Methods: This phase I multicentre, open-label, sequential-cohort, dose-escalation and dose-expansion study (NCT03886831) enrolled patients with advanced solid tumours and select haematologic malignancies. Dose-escalation study design and results were reported previously. In the dose expansion, patients with R/M ACC received recommended phase II doses of 35 or 45 mg PRT543 orally on days 1-5 of each week. Primary objectives were to establish the safety and tolerability of PRT543. Secondary objectives included efficacy., Results: Between February 2019 and May 2022, 56 patients with ACC were enrolled across 23 US sites and received either 35 mg (n = 28) or 45 mg (n = 28) of PRT543. Overall, 23% of patients experienced a grade 3 treatment-related adverse event, most commonly anaemia (16%) and thrombocytopaenia (9%). No grade 4/5 treatment-emergent adverse events were reported. Median progression-free survival was 5.9 months (95% CI: 3.8-8.3). The clinical benefit rate was 57% (95% CI: 43-70). Overall response rate (per Response Evaluation Criteria in Solid Tumours v1.1) was 2%, with 70% of patients having stable disease., Conclusion: In this analysis, PRT543 was tolerable, and the observed efficacy was limited in patients with R/M ACC., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Renata Ferrarotto: Advisory board/consulting: Ayala Pharmaceuticals, Bicara Therapeutics, Elevar Therapeutics, ExpertConnect, G1 Therapeutics, Guidepoint, Merck, Prelude Therapeutics, Regeneron, Sanofi, Remix. Royalties: Ayala Pharmaceuticals, EMD Serono, Genentech, ISA Therapeutics, Merck, Nanobiotix, Pfizer, Prelude Therapeutics, Rakuten. Paul L. Swiecicki: Grant or institutional research support: ACCRF, Ascentage Pharma; Consulting/honoraria: Elevar, Prelude Therapeutics, Remix. Dan P. Zandberg: Advisory board/consulting: Blueprint Medicines, Macrogenics, Prelude Therapeutics, Merck. Grant or institutional research support: Aduro, Astellas, AstraZeneca, Bicara, Bristol Myers Squibb, Checkmate Pharma, GlaxoSmithKline, Macrogenics, Merck, Lilly, Novasenta. Robert A. Baiocchi: Advisory board/consulting: Atara Biotherapeutics, Agenus, Viracta Therapeutics. Grant or institutional research support: Prelude Therapeutics, CODIAK Biosciences. Robert Wesolowski: Advisory board: Celcuity, Seagen. Cristina P. Rodriguez: Grant or institutional research support: AstraZeneca, Bristol Myers Squibb, Cue Biopharma, Kura, Prelude Therapeutics, Merck. Advisory board: Cue Biopharma, Coherus Biosciences. Data and safety monitoring committee: Pionyr. Meredith McKean: Grant or institutional research support: Aadi Biosciences, Accutar Biotechnology, Alpine Immune Sciences, Arcus Biosciences, Arvinas, Ascentage Pharma Group, Astellas, Bayer, Bicycle Therapeutics, BioMed Valley Discoveries, BioNTech, Dragonfly Therapeutics, EMD Serono, Epizyme, Erasca, Exelixis, Foghorn Therapeutics, G1 Therapeutics, Genentech, Gilead Sciences, GlaxoSmithKline, IDEAYA Biosciences, Ikena Oncology, ImmVira Pharma, Infinity Pharmaceuticals, Jacobio Pharmaceuticals, Kechow Pharma, Kezar Life Sciences, Kinnate BioPharma, MedImmune, Mereo BioPharma, Metabomed, Moderna, NBE Therapeutics, Nektar, Novartis, OncoC4, Oncorus, PACT Pharma, Pfizer, Plexxikon, Poseida, Prelude Therapeutics, Pyramid Biosciences, Regeneron, Sapience Therapeutics, Scholar Rock, Seattle Genetics, Synthrox, Takeda Pharmaceuticals, Teneobio, Tempest Therapeutics, Tizona Therapeutics, TMUNITY Therapeutics, TopAlliance Biosciences, Xilio. Consulting: Astellas Pharma, AstraZeneca, BicycleTX Limited, Castle Biosciences, Eisai, Ideaya Biosciences, iTeos, Moderna, Pfizer. Hyunseok Kang: Grant or institutional research support: ACCRF, Ayala Pharmaceuticals, Elevar Therapeutics, Eli Lilly, Exelixis, NeoimmuneTech, PDS Biotechnology, Prelude Therapeutics, NRG Oncology. Consulting/Honoria: Bayer, Bertis, Coherus Biosciences, MitoImmune, LG Chem, Pin Therapeutics, Remix. Varun Monga: Grant or institutional research support: Amgen, Astex Pharmaceuticals, C4 Therapeutics, Deciphera, GSK, Hutchison Pharma, Prelude Therapeutics, Rising Tide Foundation, Theradex, Trillium Therapeutics. Honoraria for lectures as part of an educational event: Gundersen Foundation, Iowa Oncology Society. Data safety monitoring board: Astex Pharmaceuticals, Forma Therapeutics. Meeting attendance: GSK, Deciphera. Rajneesh Nath: No relevant conflicts of interest to declare. Neil Palmisiano: No relevant conflicts of interest to declare. Naveen Babbar: Employee of and owns equity in Prelude Therapeutics. William Sun: Prior employee of and owns equity in Prelude Therapeutics. Glenn J. Hanna: Grant or institutional research support: ACCRF, Actuate, Elevar, Gateway for Cancer Research, Genentech, V Foundation. Consulting/honoraria: Prelude Therapeutics, Remix., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: There is increasing interest in the use of liquid biopsies, but data on longitudinal analyses of circulating tumor DNA (ctDNA) remain relatively limited. Here, we report a longitudinal ctDNA analysis of MONALEESASIA, a phase Ib trial evaluating the efficacy and safety of ribociclib plus endocrine therapy (ET) in Asian patients with hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer., Methods: MONALEESASIA enrolled premenopausal and postmenopausal Japanese and postmenopausal non-Japanese Asian patients. All patients received ribociclib with ET (letrozole, fulvestrant, or tamoxifen with goserelin). ctDNA was analyzed using a targeted next-generation sequencing panel of 572 cancer-related genes and correlated by best overall response (BOR)., Results: Five hundred seventy-four cell-free DNA samples from 87 patients were tested. The most frequently altered genes at baseline included PIK3CA (29%) and TP53 (22%). Treatment with ribociclib plus ET decreased ctDNA in most patients at the first on-treatment time point, regardless of dose or ET partner. Patients with partial response and stable disease had lower ctDNA at baseline that remained low until data cutoff if no progressive disease occurred. Most patients with progressive disease as the best response had higher ctDNA at baseline that remained high at the end of treatment. For patients with partial response and stable disease with subsequent progression, ctDNA increased towards the end of treatment in most patients, with a median lead time of 83 days (14-309 days). In some patients with BOR of partial response who experienced disease progression later, specific gene alterations and total ctDNA fraction increased; this was sometimes observed concurrently with the development of new lesions without a change in target lesion size. Patients with alterations in PIK3CA and TP53 at baseline had shorter median progression-free survival compared with patients with wild-type PIK3CA and TP53, 12.7 and 7.3 months vs 19.2 and 19.4 months, respectively (P = .016 and P = .0001, respectively)., Conclusions: Higher ctDNA levels and PIK3CA and TP53 alterations detected at baseline were associated with inferior outcomes. On-treatment ctDNA levels were associated with different patterns based on BOR. Longitudinal tracking of ctDNA may be useful for monitoring tumor status and detection of alterations with treatment implications., Trial Registration: ClinicalTrials.gov NCT02333370 . Registered on January 7, 2015., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Purpose: This analysis evaluated the genomic landscape of premenopausal patients with hormone receptor-positive and human epidermal growth factor receptor 2-negative advanced breast cancer and the association of genetic alterations with response to ribociclib in the phase III MONALEESA-7 trial., Methods: Premenopausal patients were randomly assigned 1:1 to receive endocrine therapy plus ribociclib or placebo. Plasma collected at baseline was sequenced using targeted next-generation sequencing for approximately 600 relevant cancer genes. The association of circulating tumor DNA alterations with progression-free survival (PFS) was evaluated to identify biomarkers of response and resistance to ribociclib., Results: Baseline circulating tumor DNA was sequenced in 565 patients; 489 had evidence of ≥ 1 alteration. The most frequent alterations included PIK3CA (28%), TP53 (19%), CCND1 (10%), MYC (8%), GATA3 (8%), receptor tyrosine kinases (17%), and the Chr8p11.23 locus (12%). A treatment benefit of ribociclib was seen with wild-type (hazard ratio [HR] 0.45 [95% CI, 0.33 to 0.62]) and altered (HR 0.57 [95% CI, 0.36 to 0.9]) PIK3CA . Overall, patients with altered CCND1 had shorter PFS regardless of treatment, suggesting CCND1 as a potential prognostic biomarker. Benefit with ribociclib was seen in patients with altered (HR 0.21 [95% CI, 0.08 to 0.54]) or wild-type (HR 0.52 [95% CI, 0.39 to 0.68]) CCND1 , but greater benefit was observed with altered, suggesting predictive potential of CCND1 . Alterations in TP53 , MYC , Chr8p11.23 locus, and receptor tyrosine kinases were associated with worse PFS, but ribociclib benefit was independent of alteration status., Conclusion: In this study-to our knowledge, the first large study of premenopausal patients with hormone receptor-positive and human epidermal growth factor receptor 2-negative advanced breast cancer-multiple genomic alterations were associated with poor outcome. A PFS benefit of ribociclib was observed regardless of gene alteration status, although in this exploratory analysis, a magnitude of benefits varied by alteration., Competing Interests: Aditya Bardia Consulting or Advisory Role: Novartis, Genentech, Pfizer, Spectrum Pharmaceuticals, BioTheranostics, Merck, Radius Pharma, Immunomedics, Sanofi, Puma Biotechnology, Daiichi Sankyo/Astra Zeneca, Foundation Medicine, Philips, Genentech/Roche, Radius Health, Innocrin Pharma Research Funding: Genentech, Novartis, Pfizer, Merck, Sanofi, Radius, Immunomedics, AstraZeneca/Daiichi Sankyo Open Payments Link: https://openpaymentsdata.cms.gov/physician/523675 Fei Su Employment: Novartis Stock and Other Ownership Interests: Novartis Nadia Solovieff Employment: Novartis Stock and Other Ownership Interests: Novartis Seock-Ah Im Consulting or Advisory Role: AstraZeneca, Novartis, Roche/Genentech, Eisai, Pfizer, Amgen, Hanmi, Lilly, GlaxoSmithKline, MSD, Daiichi Sankyo Research Funding: AstraZeneca, Pfizer, Roche/Genentech, Daewoong Pharmaceutical, Eisai Other Relationship: Roche Joohyuk Sohn Research Funding: MSD, Roche, Novartis, Lilly, Pfizer, Bayer, Daiichi Sankyo, AstraZeneca, GlaxoSmithKline Other Relationship: Roche Keun Seok Lee Consulting or Advisory Role: Lilly, MSD Oncology, Novartis, Roche, Pfizer Research Funding: Dong-A ST Saul Campos Gomez Consulting or Advisory Role: Roche, Bristol Myers Squibb, MSD Oncology Kyung Hae Jung Consulting or Advisory Role: Roche, AstraZeneca, Celgene, Eisai, Takeda, Novartis Marco Colleoni Research Funding: Roche Rafael Villanueva Vázquez Consulting or Advisory Role: Novartis Speakers' Bureau: Novartis, Lilly Expert Testimony: Novartis Travel, Accommodations, Expenses: Novartis, Lilly, Eisai, Daiichi Sankyo/AstraZeneca, Roche Sara Hurvitz Stock and Other Ownership Interests: Ideal Implant, ROM Tech Research Funding: Genentech/Roche, Novartis, GlaxoSmithKline, Sanofi, Pfizer, Amgen, OBI Pharma, Puma Biotechnology, Dignitana, Bayer, Biomarin, Lilly, Merrimack, Cascadian Therapeutics, Seattle Genetics, Daiichi Sankyo, Macrogenics, Ambryx, Immunomedics, Pieris Pharmaceuticals, Radius Health, Arvinas, Zymeworks, Gilead Sciences, Phoenix Molecular Designs Travel, Accommodations, Expenses: Lilly Other Relationship: Roche, Pfizer Nadia Harbeck Stock and Other Ownership Interests: West German Study Group Honoraria: Roche, Novartis, Amgen, Pfizer, AstraZeneca, Pierre Fabre, Daiichi Sankyo, Exact Sciences, MSD, Seagen Consulting or Advisory Role: Roche/Genentech, Novartis, Pfizer, Lilly, Sandoz, Daiichi Sankyo, AstraZeneca, West German Study Group, Pierre Fabre, Seagen, MSD Research Funding: Roche/Genentech, Lilly, MSD, AstraZeneca Louis Chow Research Funding: Novartis Tetiana Taran Employment: Novartis Stock and Other Ownership Interests: Novartis Karen Rodriguez Lorenc Employment: Novartis, Regeneron Stock and Other Ownership Interests: Novartis, Regeneron Naveen Babbar Employment: Novartis Stock and Other Ownership Interests: Novartis Debu Tripathy Consulting or Advisory Role: Novartis, Pfizer, GlaxoSmithKline, Genomic Health, AstraZeneca, OncoPep Research Funding: Novartis, Polyphor Travel, Accommodations, Expenses: Novartis, AstraZeneca Yen-Shen Lu Honoraria: Pfizer, Roche, Merck Sharp & Dohme, Novartis, Lilly, Eisai, Eurofarma, Daiichi Sankyo/UCB Japan, AstraZeneca Consulting or Advisory Role: Pfizer, Roche, Novartis, Lilly Research Funding: Novartis, Roche, Merck Sharp & Dohme Travel, Accommodations, Expenses: Roche, Pfizer, Eisai, Novartis No other potential conflicts of interest were reported. Aditya Bardia Consulting or Advisory Role: Novartis, Genentech, Pfizer, Spectrum Pharmaceuticals, BioTheranostics, Merck, Radius Pharma, Immunomedics, Sanofi, Puma Biotechnology, Daiichi Sankyo/Astra Zeneca, Foundation Medicine, Philips, Genentech/Roche, Radius Health, Innocrin Pharma Research Funding: Genentech, Novartis, Pfizer, Merck, Sanofi, Radius, Immunomedics, AstraZeneca/Daiichi Sankyo Open Payments Link: https://openpaymentsdata.cms.gov/physician/523675 Fei Su Employment: Novartis Stock and Other Ownership Interests: Novartis Nadia Solovieff Employment: Novartis Stock and Other Ownership Interests: Novartis Seock-Ah Im Consulting or Advisory Role: AstraZeneca, Novartis, Roche/Genentech, Eisai, Pfizer, Amgen, Hanmi, Lilly, GlaxoSmithKline, MSD, Daiichi Sankyo Research Funding: AstraZeneca, Pfizer, Roche/Genentech, Daewoong Pharmaceutical, Eisai Other Relationship: Roche Joohyuk Sohn Research Funding: MSD, Roche, Novartis, Lilly, Pfizer, Bayer, Daiichi Sankyo, AstraZeneca, GlaxoSmithKline Other Relationship: Roche Keun Seok Lee Consulting or Advisory Role: Lilly, MSD Oncology, Novartis, Roche, Pfizer Research Funding: Dong-A ST Saul Campos Gomez Consulting or Advisory Role: Roche, Bristol Myers Squibb, MSD Oncology Kyung Hae Jung Consulting or Advisory Role: Roche, AstraZeneca, Celgene, Eisai, Takeda, Novartis Marco Colleoni Research Funding: Roche Rafael Villanueva Vázquez Consulting or Advisory Role: Novartis Speakers' Bureau: Novartis, Lilly Expert Testimony: Novartis Travel, Accommodations, Expenses: Novartis, Lilly, Eisai, Daiichi Sankyo/AstraZeneca, Roche Sara Hurvitz Stock and Other Ownership Interests: Ideal Implant, ROM Tech Research Funding: Genentech/Roche, Novartis, GlaxoSmithKline, Sanofi, Pfizer, Amgen, OBI Pharma, Puma Biotechnology, Dignitana, Bayer, Biomarin, Lilly, Merrimack, Cascadian Therapeutics, Seattle Genetics, Daiichi Sankyo, Macrogenics, Ambryx, Immunomedics, Pieris Pharmaceuticals, Radius Health, Arvinas, Zymeworks, Gilead Sciences, Phoenix Molecular Designs Travel, Accommodations, Expenses: Lilly Other Relationship: Roche, Pfizer Nadia Harbeck Stock and Other Ownership Interests: West German Study Group Honoraria: Roche, Novartis, Amgen, Pfizer, AstraZeneca, Pierre Fabre, Daiichi Sankyo, Exact Sciences, MSD, Seagen Consulting or Advisory Role: Roche/Genentech, Novartis, Pfizer, Lilly, Sandoz, Daiichi Sankyo, AstraZeneca, West German Study Group, Pierre Fabre, Seagen, MSD Research Funding: Roche/Genentech, Lilly, MSD, AstraZeneca Louis Chow Research Funding: Novartis Tetiana Taran Employment: Novartis Stock and Other Ownership Interests: Novartis Karen Rodriguez Lorenc Employment: Novartis, Regeneron Stock and Other Ownership Interests: Novartis, Regeneron Naveen Babbar Employment: Novartis Stock and Other Ownership Interests: Novartis Debu Tripathy Consulting or Advisory Role: Novartis, Pfizer, GlaxoSmithKline, Genomic Health, AstraZeneca, OncoPep Research Funding: Novartis, Polyphor Travel, Accommodations, Expenses: Novartis, AstraZeneca Yen-Shen Lu Honoraria: Pfizer, Roche, Merck Sharp & Dohme, Novartis, Lilly, Eisai, Eurofarma, Daiichi Sankyo/UCB Japan, AstraZeneca Consulting or Advisory Role: Pfizer, Roche, Novartis, Lilly Research Funding: Novartis, Roche, Merck Sharp & Dohme Travel, Accommodations, Expenses: Roche, Pfizer, Eisai, Novartis No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Purpose: The prognostic and predictive value of intrinsic subtypes in hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer treated with endocrine therapy and ribociclib (RIB) is currently unknown. We evaluated the association of intrinsic subtypes with progression-free survival (PFS) in the MONALEESA trials., Methods: A retrospective and exploratory PAM50-based analysis of tumor samples from the phase III MONALEESA-2, MONALEESA-3, and MONALEESA-7 trials was undertaken. The prognostic relationship of PAM50-based subtypes with PFS and risk of disease progression by subtype and treatment were evaluated using a multivariable Cox proportional hazards model, adjusting for age, prior chemotherapy, performance status, visceral disease, bone-only metastases, histological grade, number of metastatic sites, prior endocrine therapy, and de novo metastatic disease., Results: Overall, 1,160 tumors from the RIB (n = 672) and placebo (n = 488) cohorts were robustly profiled. Subtype distribution was luminal A (LumA), 46.7%; luminal B (LumB), 24.0%; normal-like, 14.0%; HER2-enriched (HER2E), 12.7%; and basal-like, 2.6% and was generally consistent across treatment arms and trials. The associations between subtypes and PFS were statistically significant in both arms ( P < .001). The risks of disease progression for LumB, HER2E, and basal-like subtypes were 1.44, 2.31, and 3.96 times higher compared with those for LumA, respectively. All subtypes except basal-like demonstrated significant PFS benefit with RIB. HER2E (hazard ratio [HR], 0.39; P < .0001), LumB (HR, 0.52; P < .0001), LumA (HR, 0.63; P = .0007), and normal-like (HR, 0.47; P = .0005) subtypes derived benefit from RIB. Patients with basal-like subtype (n = 30) did not derive benefit from RIB (HR, 1.15; P = .77)., Conclusion: In this retrospective exploratory analysis of hormone receptor-positive and human epidermal growth factor receptor 2-negative advanced breast cancer, each intrinsic subtype exhibited a consistent PFS benefit with RIB, except for basal-like., Competing Interests: Aleix PratHonoraria: Nanostring Technologies, Bristol Myers Squibb, Novartis, Merck Sharp & Dohme, Pfizer, Roche, Lilly, Oncolytics Biotech, Amgen, Daiichi Sankyo, PUMA, Boehringer Anwesha ChaudhuryEmployment: Novartis Nadia SolovieffEmployment: NovartisStock and Other Ownership Interests: Novartis Nuria ChicTravel, Accommodations, Expenses: Novartis Agnes LteifEmployment: NovartisStock and Other Ownership Interests: Novartis Tetiana TaranEmployment: NovartisStock and Other Ownership Interests: Novartis Naveen BabbarEmployment: NovartisStock and Other Ownership Interests: Novartis Fei SuEmployment: NovartisStock and Other Ownership Interests: NovartisNo other potential conflicts of interest were reported.

Hepatic hemangioma is a commonly encountered benign vascular tumour of liver during infancy. Acquired hypothyroidism is one of the rare manifestation of this entity. We report a 4-month-old infant born to a diabetic mother who developed acquired hypothyroidism not responding to treatment due to multiple hepatic hemangiomas. The mechanism behind is increased type 3 deiodinase activity due to hemangiomas of liver, which catalyses conversion of T4 to rT3 and T3 to T2. Hemangiomas were successfully treated with propranolol which lead to its regression and ultimately resulted in euthyroidism. Hence, screening for hepatic hemangioma should be done in all cases of congenital hypothyroidism not responding to thyroxine treatment. We also propose early initiation of treatment of hemangiomas with propranolol as the first line therapy to prevent growth retardation and intellectual loss., Competing Interests: There are no conflicts of interest., (Copyright: © 2020 Journal of Family Medicine and Primary Care.)

Background: The delay in the diagnosis and treatment initiation of patients with MDR-TB worsens individual prognosis and increases the risk of disease transmission in the community. These delays have been attributed to delay in treatment-seeking by the patient and shifting to multiple healthcare facilities before being tested and diagnosed through India's National Tuberculosis Elimination Program (NTEP)., Objective: to identify treatment pathways in patients with MDR-TB from the time of onset of symptoms and treatment seeking until diagnosis at a PMDT site and subsequent treatment initiation. We also compared these characteristics with those of patients with DS-TB., Methods: We recruited a total of 168 patients with MDR-TB and DS-TB each, in Delhi. Data were analyzed using IBM SPSS Version 25., Results: The mean (SD) patient delay for initial treatment-seeking was 20.9 (15.9) days in patients with MDR-TB, and 16.1 (17.1) days in patients with DS-TB (p < 0.001). The median time from visit to the first healthcare facility (HCF) until confirmation of MDR-TB diagnosis was 78.5 days, and until treatment initiation was 102.5 days. Among patients with DS-TB, the time interval from a visit to the first HCF until the initiation of ATT-DOTS was 61.5 days.. Patients diagnosed with DS-TB, whose first source of treatment was a private facility (n = 49), reported a significant delay in the initiation of ATT-DOTS (p < 0.001)., Conclusions: Despite the introduction of universal drug sensitivity testing in individuals having presumptive MDR-TB, a significant delay in the diagnosis and initiation of effective MDR-TB treatment persists as a major public health challenge in India., (Copyright © 2020 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

X-linked lissencephaly, absent corpus callosum, and epilepsy of neonatal onset with ambiguous genitalia are the classical features of XLAG syndrome and as of now very few cases have been reported in the literature. In this study, we present the case of XLAG syndrome who presented in neonatal period with refractory seizures and ambiguous genitalia. MRI brain showed abnormal gyral pattern with smooth broad gyri suggestive of Lissencephaly and agenesis of corpus callosum. Our index case survived for only 25 days. Early suspicion, genetic counselling, and prenatal radiological work-up of such cases will reduce further burden on the family., Competing Interests: There are no conflicts of interest., (Copyright: © 2020 Journal of Family Medicine and Primary Care.)

Background: Combined targeting of CDK4/6 and ER is now the standard of care for patients with advanced ER+/HER2- breast cancer. However, acquired resistance to these therapies frequently leads to disease progression. As such, it is critical to identify the mechanisms by which resistance to CDK4/6-based therapies is acquired and also identify therapeutic strategies to overcome resistance., Methods: In this study, we developed and characterized multiple in vitro and in vivo models of acquired resistance to CDK4/6-based therapies. Resistant models were screened by reverse phase protein array (RPPA) for cell signaling changes that are activated in resistance., Results: We show that either a direct loss of Rb or loss of dependence on Rb signaling confers cross-resistance to inhibitors of CDK4/6, while PI3K/mTOR signaling remains activated. Treatment with the p110α-selective PI3K inhibitor, alpelisib (BYL719), completely blocked the progression of acquired CDK4/6 inhibitor-resistant xenografts in the absence of continued CDK4/6 inhibitor treatment in models of both PIK3CA mutant and wild-type ER+/HER2- breast cancer. Triple combination therapy against PI3K:CDK4/6:ER prevented and/or delayed the onset of resistance in treatment-naive ER+/HER2- breast cancer models., Conclusions: These data support the clinical investigation of p110α-selective inhibitors of PI3K, such as alpelisib, in patients with ER+/HER2- breast cancer who have progressed on CDK4/6:ER-based therapies. Our data also support the investigation of PI3K:CDK4/6:ER triple combination therapy to prevent the onset of resistance to the combination of endocrine therapy plus CDK4/6 inhibition.

Background & Objectives: The increase in the burden of multidrug-resistant tuberculosis (MDR-TB) is a matter of grave concern. The present study was undertaken to describe MDR-TB treatment outcome trends in Delhi and their epidemiological correlates, to assess the adequacy of treatment records and to also generate evidence towards influencing and improving practices related to the MDR-TB control programme., Methods: A retrospective record-based study (2009-2014) was conducted in three major drug resistance TB treatment centres of Delhi. Treatment outcomes and adverse effects were extracted from the existing programme records including patients' treatment cards and laboratory registers., Results: A total of 2958 MDR-TB patients were identified from the treatment cards, of whom 1749 (59.12%) were males. The mean (±standard deviation) age was 30.56±13.5 years. Favourable treatment outcomes were reported in 1371 (53.28%) patients, but they showed a declining trend during the period of observation. On binomial logistic regression analysis, patients with age ≥35 yr, male sex and undernourishment (body mass index <18.5) at the time of treatment initiation had a significantly increased likelihood of unfavourable MDR-TB treatment outcome (P <0.001)., Interpretation & Conclusions: The study showed an increasing burden of MDR-TB patients, especially in the young population with increased risk of transmission posing a major challenge in achieving TB elimination targets., Competing Interests: None

Related neurological adverse effects to metronidazole are rarely encountered in clinical practice despite its wide use as an antibacterial or antiparasitic agent. The neurotoxicity is not dose-dependent and is fully reversible with discontinuation of the drug. We describe a young man who was receiving metronidazole for an amoebic liver abscess and developed encephalopathy and seizures. Brain magnetic resonance imaging showed characteristic bilateral symmetrical cerebellar dentate hyperintensities.

Purpose: Addition of alpelisib to fulvestrant significantly extended progression-free survival in PIK3CA -mutant, hormone receptor-positive (HR + ) advanced/metastatic breast cancer in the phase III SOLAR-1 study. The combination of alpelisib and letrozole also had promising activity in phase I studies of HR + advanced/metastatic breast cancer. NEO-ORB aimed to determine whether addition of alpelisib to letrozole could increase response rates in the neoadjuvant setting. Patients and Methods: Postmenopausal women with HR + , human epidermal growth factor receptor 2-negative, T1c-T3 breast cancer were assigned to the PIK3CA -wild-type or PIK3CA -mutant cohort according to their tumor PIK3CA status, and randomized (1:1) to 2.5 mg/day letrozole with 300 mg/day alpelisib or placebo for 24 weeks. Primary endpoints were objective response rate (ORR) and pathologic complete response (pCR) rate for both PIK3CA cohorts., Results: In total, 257 patients were assigned to letrozole plus alpelisib (131 patients) or placebo (126 patients). Grade ≥3 adverse events (≥5% of patients) in the alpelisib arm were hyperglycemia (27%), rash (12%), and maculo-papular rash (8%). The primary objective was not met; ORR in the alpelisib versus placebo arm was 43% versus 45% and 63% versus 61% in the PIK3CA -mutant and wild-type cohorts, respectively. pCR rates were low in all groups. Decreases in Ki-67 were similar across treatment arms and cohorts. In PIK3CA -mutant tumors, alpelisib plus letrozole treatment induced a greater decrease in phosphorylated AKT versus placebo plus letrozole., Conclusions: In contrast to initial results in advanced/metastatic disease, addition of alpelisib to 24-week neoadjuvant letrozole treatment did not improve response in patients with HR + early breast cancer., (©2019 American Association for Cancer Research.)

Background: There are over 12,000 chemists registered in the capital city, Delhi to support patient health needs. A study was conducted to improve the tuberculosis (TB) notification rates as conceptualized by the Revised National Tuberculosis Control Program (RNTCP). As part of the end TB mission, the feasibility of capturing data of TB patients coming to buy anti-TB drugs at the licensee level (chemists and drug shop owners) in Central Delhi area was assessed., Materials and Methods: The prospective study was conducted from July 2017 to March 2018. TB notification through a paper-based system and self-notification through online mode were the operational modality used for engagement with chemists. A team of paramedical workers was deployed for data collection from those pharmacists who chose to notify through the paper mode. Self-notification through online mode was through the RNTCP's NIKSHAY web-based reporting platform., Results: From the 330 chemists sensitized, 871 TB notifications were received during the study. Younger age groups comprised a majority of these cases with 198 (37.5%) from 21 to 30 years and 122 (23.1%) from 11 to 20 years. By the end of six visits, 28 (46%) of the 61 pharmacies that were eventually successfully sensitized had started returning the Folio cards with filled patient details. A total of 581 (66.6%) prescriptions received by the pharmacists were from government hospitals. The annual TB case notification in Central Delhi showed a significant increase from 271 TB patients/100,000 population to 871 TB patients/100,000 population during the study period when compared with expected trends in the past year ( P < 0.05)., Conclusion: Self-notification of TB engenders successful TB notifications from chemists. This progenitor approach to TB notification in the capital emphasizes the need to categorize pharmacists as an independent private care provider for improving TB notification across high-burden settings., Competing Interests: There are no conflicts of interest.

Metastasis is the main cause of death for patients with breast cancer. Many studies have characterized the genomic landscape of breast cancer during its early stages. However, there is evidence that genomic alterations are acquired during the evolution of cancers from their early to late stages, and that the genomic landscape of early cancers is not representative of that of lethal cancers 1-7 . Here we investigated the landscape of somatic alterations in 617 metastatic breast cancers. Nine driver genes (TP53, ESR1, GATA3, KMT2C, NCOR1, AKT1, NF1, RIC8A and RB1) were more frequently mutated in metastatic breast cancers that expressed hormone receptors (oestrogen and/or progesterone receptors; HR + ) but did not have high levels of HER2 (HER2 - ; n = 381), when compared to early breast cancers from The Cancer Genome Atlas. In addition, 18 amplicons were more frequently observed in HR + /HER2 - metastatic breast cancers. These cancers showed an increase in mutational signatures S2, S3, S10, S13 and S17. Among the gene alterations that were enriched in HR + /HER2 - metastatic breast cancers, mutations in TP53, RB1 and NF1, together with S10, S13 and S17, were associated with poor outcome. Metastatic triple-negative breast cancers showed an increase in the frequency of somatic biallelic loss-of-function mutations in genes related to homologous recombination DNA repair, compared to early triple-negative breast cancers (7% versus 2%). Finally, metastatic breast cancers showed an increase in mutational burden and clonal diversity compared to early breast cancers. Thus, the genomic landscape of metastatic breast cancer is enriched in clinically relevant genomic alterations and is more complex than that of early breast cancer. The identification of genomic alterations associated with poor outcome will allow earlier and better selection of patients who require the use of treatments that are still in clinical trials. The genetic complexity observed in advanced breast cancer suggests that such treatments should be introduced as early as possible in the disease course.

The aim of this research was to valorize onion skins, an under-utilized agricultural by-product, into pectic oligosaccharides (POS), compounds with potential health benefits. To achieve high hydrolysis performance with the multi-activity enzyme Viscozyme L, an innovative approach was investigated based on a cross-flow continuous membrane enzyme bioreactor (EMR). The influence of the various process conditions (residence time, enzyme concentration, substrate concentration) was investigated on productivity and yield. The composition of the POS mixtures in terms of mono- and oligosaccharides was assessed at the molecular level. At optimized conditions, a stable POS production with 22.0g/L/h volumetric productivity and 4.5g/g POS/monosaccharides was achieved. Compared to previous results obtained in batch for the enzyme Viscozyme L, EMR provided a 3-5× higher volumetric productivity for the smallest POS. Moreover, it gave competitive results even when compared to batch production with a pure endo-galacturonase enzyme, demonstrating its feasibility for efficient POS production., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Sugar beet pulp pectin is an attractive source for the production of pectic oligosaccharides, an emerging class of potential prebiotics. The main aim of the present work was to investigate a new process allowing to produce pectic oligosaccharides in a continuous way by means of a cross flow enzyme membrane reactor while using a low-cost crude enzyme mixture (viscozyme). Preliminary experiments in batch and semi-continuous setups allowed to identify suitable enzyme concentrations and assessing filtration suitability. Then, in continuous experiments in the enzyme membrane reactor, residence time and substrate loading were further optimized. The composition of the obtained oligosaccharide mixtures was assessed at the molecular level for the most promising conditions and was shown to be dominated by condition-specific arabinans, rhamnogalacturonans, and galacturonans. A continuous and stable production was performed for 28.5 h at the optimized conditions, obtaining an average pectic oligosaccharide yield of 82.9 ± 9.9% (w/w), a volumetric productivity of 17.5 ± 2.1 g/L/h, and a specific productivity of 8.0 ± 1.0 g/g E/h. This work demonstrated for the first time the continuous and stable production of oligosaccharide mixtures from sugar beet pulp using enzyme membrane reactor technology in a setup suitable for upscaling.

Acquired factor VII (FVII) deficiency in the absence of vitamin K deficiency, oral, synthetic liver dysfunction, or DIC is rare, with only a handful of cases published in literature. Congenital deficiency of FVII is well known but, little is known about secondary FVII deficiency and its management. The knowledge of this syndrome should avoid false diagnosis of congenital factor VII deficiency. Here, we present the report of a young woman who presented with pyelonephritis, anaemia, prolonged prothrombin time and normal activated partial prothrombin time (aPTT). She was diagnosed to have acquired FVII deficiency and this was the first such case with pyelonephritis.

Pectin oligosaccharides (POS) have been indicated as a new class of potential prebiotic compounds, which can be produced from pectin-rich food byproducts. In the present study, different technological means of POS production were explored to produce tailor-made POS mixtures starting from sugar beet pulp. The overall POS production process consisted of two steps: the extraction of pectin and the hydrolysis of pectin to tailored POS by combined hydrolysis/fractionation approaches. Different extraction as well as hydrolysis and fractionation methodologies were applied. The obtained POS were characterized for their total galacturonic acid content and, at a deeper level, using a HILIC-ESI/MS methodology, for the POS structure and composition. The composition of POS fractions was studied as a function of the technology used to obtain them. Finally, the potential prebiotic properties of the POS mixtures obtained were thoroughly explored by several in vitro experiments aimed at detecting lactic acid bacteria (LAB) stimulation by POS fractions. Several fractions were very efficient in stimulation, in a species-dependent manner. The overall best fractions were in general those rich in arabinans having a low degree of polymerization, obtained from the enzymatic extraction of biomass and subsequent fractionation with low-medium molecular weight cut-off. Quite interestingly, no POS fraction was able to stimulate pathogenic E. coli strains. The data reported here clearly indicate the possibility to obtain diverse fractions with different prebiotic properties starting from the same biomass, and outline clear potential for POS obtained from sugar beet pulp with the appropriate technology to act as prebiotic compounds.

Pectic oligosaccharides (POS) have been indicated as novel candidate prebiotics. Traditionally, POS are produced from pectin-rich by-products using a two-step process involving extraction of the pectin, followed by its hydrolysis into POS. A one-step approach, in which the POS is directly produced from the raw material, might provide a more efficient alternative. Thus, the main aim of this paper was to investigate a one-step enzymatic hydrolysis approach to directly produce POS from sugar beet pulp (SBP). The POS yield was investigated as a function of the process parameters, as well as raw material characteristics. A statistically-based response surface methodology, using a central composite design was applied, to investigate the individual as well as the combined influences of the diverse parameters. The model was confirmed by a validation experiment, carried out at 135 g/l substrate concentration, 0.75 FPU/g SBP enzyme concentration, 0.8 mm particle size and 3 h hydrolysis time. Under these conditions, a POS-rich hydrolysate was obtained, containing rhamnose, arabinose, galactose, xylose and galacturonic acid, at 0.9, 15.2, 5.1, 1.4, and 13.2 g/l, respectively, enzymes were added each at 20 FPU/g dry matter (DM).

Pectin containing agricultural by-products are potential sources of a new class of prebiotics known as pectic oligosaccharides (POS). In general, pectin is made up of homogalacturonan (HG, α-1,4-linked galacturonic acid monomers) and rhamnogalacturonan (RG, alternate galacturonic acid and rhamnose backbone with neutral side chains). Controlled hydrolysis of pectin containing agricultural by-products like sugar beet, apple, olive and citrus by chemical, enzymatic and hydrothermal can be used to produce oligo-galacturonides (GalpOS), galacto-oligosaccharides (GalOS), rhamnogalacturonan-oligosaccharides (RGOS), etc. However, extensive research is needed to establish the role of POS, both as a prebiotic as well as therapeutic agent. This review comprehensively covers different facets of POS, including the nature and chemistry of pectin and POS, potential agricultural residual sources of pectin, pre-treatment methods for facilitating selective extraction of pectin, identification and characterization of POS, health benefits and important applications of POS in food and feed. This review has been compiled to establish a platform for future research in the purification and characterization of POS and for in vivo and in vitro studies of important POS, so that they could be commercially exploited.

Onion skins are evaluated as a new raw material for the enzymatic production of pectic oligosaccharides (POS) with a targeted degree of polymerization (DP). The process is based on a two-stage process consisting of a chelator-based crude pectin extraction followed by a controlled enzymatic hydrolysis. Treatment of the extracted crude onion skin's pectin with various enzymes (EPG-M2, Viscozyme and Pectinase) shows that EPG-M2 is the most appropriate enzyme for tailored POS production. The experiments reveal that the highest amount of DP2 and DP3 is obtained at a time scale of 75-90min with an EPG-M2 concentration of 26IU/mL. At these conditions the production amounts 2.5-3.0% (w/w) d.m for DP2 and 5.5-5.6% (w/w) d.m for DP3 respectively. In contrast, maximum DP4 production of 5.2-5.5% (w/w) d.m. is obtained with 5.2IU/mL at a time scale of 15-30min. Detailed LC-MS analysis reveals the presence of more methylated oligomers compared to acetylated forms in the digests., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Colorectal cancer (CRC) testing programs reduce mortality; however, approximately 40% of the recommended population who should undergo CRC testing does not. Early colon cancer detection in patient populations ineligible for testing, such as the elderly or those with significant comorbidities, could have clinical benefit. Despite many attempts to identify individual protein markers of this disease, little progress has been made. Targeted mass spectrometry, using multiple reaction monitoring (MRM) technology, enables the simultaneous assessment of groups of candidates for improved detection performance., Materials and Methods: A multiplex assay was developed for 187 candidate marker proteins, using 337 peptides monitored through 674 simultaneously measured MRM transitions in a 30-minute liquid chromatography-mass spectrometry analysis of immunodepleted blood plasma. To evaluate the combined candidate marker performance, the present study used 274 individual patient blood plasma samples, 137 with biopsy-confirmed colorectal cancer and 137 age- and gender-matched controls. Using 2 well-matched platforms running 5 days each week, all 274 samples were analyzed in 52 days., Results: Using one half of the data as a discovery set (69 disease cases and 69 control cases), the elastic net feature selection and random forest classifier assembly were used in cross-validation to identify a 15-transition classifier. The mean training receiver operating characteristic area under the curve was 0.82. After final classifier assembly using the entire discovery set, the 136-sample (68 disease cases and 68 control cases) validation set was evaluated. The validation area under the curve was 0.91. At the point of maximum accuracy (84%), the sensitivity was 87% and the specificity was 81%., Conclusion: These results have demonstrated the ability of simultaneous assessment of candidate marker proteins using high-multiplex, targeted-mass spectrometry to identify a subset group of CRC markers with significant and meaningful performance., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The influence of different extraction methodologies was assessed on the composition of both neutral (arabinose, rhamnose, galactose) and acidic (galacturonic acid) pectic polysaccharides obtained from four agro-industrial residues, namely, berry pomace (BP), onion hulls (OH), pressed pumpkin (PP), and sugar beet pulp (SBP). For acidic pectic polysaccharides, the extraction efficiency was obtained as BP (nitric acid-assisted extraction, 2 h, 62.9%), PP (enzymatic-assisted extraction, 12 h, 75.0%), SBP (enzymatic-assisted extraction, 48 h, 89.8%; and nitric acid-assisted extraction, 4 h, 76.5%), and OH (sodium hexametaphosphate-assisted extraction, 0.5 h, 100%; and ammonium oxalate-assisted extraction, 0.5 h, 100%). For neutral pectic polysaccharides, the following results were achieved: BP (enzymatic-assisted extraction, 24 h, 85.9%), PP (nitric acid-assisted extraction, 6 h, 82.2%), and SBP (enzymatic assisted extraction, 48 h, 97.5%; and nitric acid-assisted extraction, 4 h, 83.2%). On the basis of the high recovery of pectic sugars, SBP and OH are interesting candidates for the further purification of pectin and production of pectin-derived products.

Introduction: In India, almost half of all patients with tuberculosis (TB) seek care in the private sector as the first point of care. The national programme is unable to support such TB patients and facilitate effective treatment, as there is no information on TB and Multi or Extensively Drug Resistant TB (M/XDR-TB) diagnosis and treatment in private sector., Objective: To improve this situation, Government of India declared TB a notifiable disease for establishing TB surveillance system, to extend supportive mechanism for TB treatment adherence and standardised practices in the private sector. But TB notification from the private sector is a challenge and still a lot needs to be done to accelerate TB notification., Methods: Delhi State TB Control Programme had taken initiatives for improving notification of TB cases from the private sector in 2014. Key steps taken were to constitute a state level TB notification committee to oversee the progress of TB notification efforts in the state and direct 'one to one' sensitisation of private practitioners (PPs) (in single PP's clinic, corporate hospitals and laboratories) by the state notification teams with the help of available tools for sensitising the PP on TB notification - TB Notification Government Order, Guidance Tool for TB Notification and Standards of TB Care in India., Results: As a result of focussed state level interventions, without much external support, there was an accelerated notification of TB cases from the private sector. TB notification cases from the private sector rose from 341 (in 2013) to 4049 (by the end of March 2015)., Conclusion: Active state level initiatives have led to increase in TB case notification., (Copyright © 2016 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

The growing interest in the substitution of synthetic food antioxidants by natural ones has fostered research in identifying new low-cost antioxidants having commercial potential. Fruits such as mango, banana, and those belonging to the citrus family leave behind a substantial amount of residues in the form of peels, pulp, seeds, and stones. Due to lack of infrastructure to handle a huge quantity of available biomass, lack of processing facilities, and high processing cost, these residues represent a major disposal problem, especially in developing countries. Because of the presence of phenolic compounds, which impart nutraceutical properties to fruit residues, such residues hold tremendous potential in food, pharmaceutical, and cosmetic industries. The biological properties such as anticarcinogenicity, antimutagenicity, antiallergenicity, and antiageing activity have been reported for both natural as well as synthetic antioxidants. Special attention is focused on extraction of bioactive compounds from inexpensive or residual sources. The purpose of this review is to characterize different phenolics present in the fruit residues, discuss the antioxidant potential of such residues and the assays used in determination of antioxidant properties, discuss various methods for efficient extraction of the bioactive compounds, and highlight the importance of fruit residues as potential nutraceutical resources and biopreservatives.

Dried residues from four different vegetables, viz. pea pod (pp), cauliflower waste (CW), potato peel (PP) and tomato peel (TP) were extracted using four solvents i.e., hexane, chloroform, ethyl acetate and methanol. Among the four solvents, methanolic extracts showed the highest total phenolic content (TPC) for all the four vegetable residues. Methanolic extracts were evaluated for antioxidant activities using diphenylpicryl-hydrazyl (DPPH) and reducing power assay. Tomato peel extract showed highest phenolic content of 21.0 mg GAE/g-dw and 80.8 % DPPH free radical scavenging ability, whereas potato peel extract had a low phenolic content, and it also showed the least antioxidant activity among the residues examined in this study. Total phenolic content and DPPH free radical scavenging activity in pea pods and cauliflower waste were 13.6 mg GAE/g-dw and 72 % and 9.2 mg GAE/g-dw and 70.7 %, respectively. The coefficient of determination (r(2)) for correlation between TPC and reducing power, DPPH and TPC, DPPH and reducing power for all extracts was 0.85, 0.91and 0.87, respectively, suggesting an important role of phenolics in imparting antioxidant ability. Extracts from vegetables residues therefore represent a significant source of phenolic antioxidants for use as nutraceuticals or biopreservatives.

Cellulolytic enzyme production by newly isolated Aspergillus niger HN-1 was statistically optimized using Plackett-Burman and central composite design (CCD). Optimum concentrations of 2, 0.40, 0.01, and 0.60 g L (-1) for KH2PO4, urea, trace elements solution, and CaCl2·2H2O, respectively, were suggested by Design-Expert software. The two-stage optimization process led to a 3- and 2-fold increases in the filter paper cellulase (FP) and β-glucosidase activities, respectively. FP, β-glucosidase, endoglucanase, exopolygalaturonase, cellobiohydrolase, xylanase, α-l-arabinofuranosidase, β-xylosidase, and xylan esterase activities of 36.7 ± 1.54 FPU gds(-1), 252.3 ± 7.4 IU gds(-1), 416.3 ± 22.8 IU gds(-1), 111.2 ± 5.4 IU gds(-1), 8.9 ± 0.50 IU gds(-1), 2593.5 ± 78.9 IU gds(-1), 79.4 ± 4.3 IU gds(-1), 180.8 ± 9.3 IU gds(-1), and 288.7 ± 11.8 IU gds(-1), respectively, were obtained through solid-state fermentation during the validation studies. Hydrolysis of alkali-treated rice straw with crude cellulases resulted in about 84% glucan to glucose, 89% xylan to xylose, and 91% arabinan to arabinose conversions, indicating potential for biomass hydrolysis by the crude cellulase consortium obtained in this study.

In this study, simultaneous saccharification and fermentation (SSF) was employed to produce ethanol from 1% sodium hydroxide-treated rice straw in a thermostatically controlled glass reactor using 20 FPU gds⁻¹ cellulase, 50 IU gds⁻¹ β-glucosidase, 15 IU gds⁻¹ pectinase and a newly isolated thermotolerant Pichia kudriavzevii HOP-1 strain. Scanning electron micrograph images showed that the size of the P. kudriavzevii cells ranged from 2.48 to 6.93 μm in diameter while the shape of the cells varied from oval, ellipsoidal to elongate. Pichia kudriavzevii cells showed extensive pseudohyphae formation after 5 days of growth and could assimilate sugars like glucose, sucrose, galactose, fructose, and mannose but the cells could not assimilate xylose, arabinose, cellobiose, raffinose, or trehalose. In addition, the yeast cells could tolerate up to 40% glucose and 5% NaCl concentrations but their growth was inhibited at 1% acetic acid and 0.01% cyclohexamide concentrations. Pichia kudriavzevii produced about 35 and 200% more ethanol than the conventional Saccharomyces cerevisiae cells at 40 and 45°C, respectively. About 94% glucan in alkali-treated rice straw was converted to glucose through enzymatic hydrolysis within 36 h. Ethanol concentration of 24.25 g l⁻¹ corresponding to 82% theoretical yield on glucan basis and ethanol productivity of 1.10 g l⁻¹ h⁻¹ achieved using P. kudriavzevii during SSF hold promise for scale-up studies. An insignificant amount of glycerol and no xylitol was produced during SSF. To the best of our knowledge, this is the first study reporting ethanol production from any lignocellulosic biomass using P. kudriavzevii.

Dried, ground, and hydrothermally pretreated Kinnow mandarin (Citrus reticulata) waste was used to produce ethanol via simultaneous saccharification and fermentation (SSF). Central composite design was used to optimize cellulase and pectinase concentrations, temperature, and time for SSF. The D-limonene concentration determined with high-performance liquid chromatography (HPLC) for fresh, dried, and pretreated biomass was 0.76%, 0.32%, and 0.09% (v/w), respectively. Design Expert software suggested that the first-order effect of all four factors and the second-order effect of cellulase and pectinase concentrations were significant for ethanol production. The validation experiment using 6 FPU gds(-1) cellulase and 60 IU gds(-1) pectinase at 37 °C for 12 h in a laboratory batch fermenter resulted in ethanol concentration and productivity of 42 g L(-1) and 3.50 g L(-1) h(-1), respectively. Experiments using optimized parameters resulted in an ethanol concentration similar to that predicted by the model equation and also helped reduce fermentation time., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Increased polyamine synthesis and inflammation have long been associated with intraepithelial neoplasia, which are risk factors for cancer development in humans. Targeting polyamine metabolism (by use of polyamine synthesis inhibitors or polyamine catabolism activators) and inflammation (by use of nonsteroidal anti-inflammatory drugs) has been studied for many cancers, including colon, prostate, and skin. Genetic epidemiology results indicate that a genetic variant associated with the expression of a polyamine biosynthetic gene is associated with risk of colon and prostate cancers. A clinical trial of difluoromethylornithine (DFMO), a selective inhibitor of polyamine synthesis, showed that the 1 year treatment duration reduced prostate volume and serum prostate-specific antigen doubling time in men with a family history of prostate cancer. A second, clinical trial of DFMO in combination with sulindac, a NSAID in patients with prior colon polyps found that the 3-year treatment was associated with a 70% reduction of all, and over a 90% reduction of advanced and/or multiple metachronous colon adenomas. In this chapter, we discuss that similar combination prevention strategies of targeting polyamines and inflammation can be effective in reducing risk factors associated with the development of human cancers.

Inflammation has been implicated in the development of many human epithelial cancers, including those of the stomach, lung, colon, and prostate. Tumor necrosis factor-alpha (TNF-alpha) is a potent pleiotropic, proinflammatory cytokine produced by many cells in response to injury and inflammation. Here, we show that TNF-alpha exposure results in increased production of reactive oxygen species (ROS), with a concomitant increase in the production of 8-oxo-deoxyguanosine, a marker for oxidative DNA damage, in human lung bronchial epithelial cells. The source of the ROS in TNF-alpha-treated cells was determined by both pharmacologic and small interfering RNA (siRNA) strategies to be spermine oxidase (SMO/PAOh1). SMO/PAOh1 oxidizes spermine into spermidine, 3-aminopropanal, and H(2)O(2). Inhibition of TNF-alpha-induced SMO/PAOh1 activity with MDL 72,527 or with a targeted siRNA prevented ROS production and oxidative DNA damage. Further, similar induction in SMO/PAOh1 is observed with treatment of another inflammatory cytokine, interleukin-6. The data are consistent with a model that directly links inflammation and DNA damage through the production of H(2)O(2) by SMO/PAOh1. Further, these results suggest a common mechanism by which inflammation from multiple sources can lead to the mutagenic changes necessary for the development and progression of epithelial cancers.

Tumor necrosis factor alpha (TNFalpha) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNFalpha are due to its ability to activate multiple signal transduction pathways, including nuclear factor kappaB (NFkappaB), which plays critical roles in cell proliferation and survival. TNFalpha displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNFalpha can lead to the induction of NFkappaB signaling with a concomitant increase in spermidine/spermine N(1)-acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a rate-limiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IkappaBalpha protein led to the suppression of SSAT induction by TNFalpha in these cells, thereby substantiating a role of NFkappaB in the induction of SSAT by TNFalpha. SSAT promoter deletion constructs led to the identification of three potential NFkappaB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFkappaB response elements play an important role in the regulation of SSAT in response to TNFalpha. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFkappaB signaling pathway in non-small cell lung cancer cells.

Epidemiological, experimental and clinical results suggest that aspirin and other NSAIDs (non-steroidal anti-inflammatory drugs) inhibit the development of colon cancer. It has been shown that the NSAID sulindac induces apoptosis and suppresses carcinogenesis, in part, by a mechanism leading to the transcriptional activation of the gene encoding SSAT (spermidine/spermine N1-acetyltransferase), a rate-limiting enzyme in polyamine catabolism. In the present study, we show that a variety of NSAIDs, including aspirin, sulindac, ibuprofen and indomethacin, can induce SSAT gene expression in Caco-2 cells. Aspirin, at physiological concentrations, can induce SSAT mRNA via transcriptional initiation mechanisms. This induction leads to increased SSAT protein levels and enzyme activity. Promoter deletion analysis of the 5' SSAT promoter-flanking region led to the identification of two NF-kappaB (nuclear factor kappaB) response elements. Electrophoretic mobility-shift assays showed binding of NF-kappaB complexes at these sequences after aspirin treatment. Aspirin treatment led to the activation of NF-kappaB signalling and increased binding at these NF-kappaB sites in the SSAT promoter, hence providing a potential mechanism for the induction of SSAT by aspirin in these cells. Aspirin-induced SSAT ultimately leads to a decrease in cellular polyamine content, which has been associated with decreased carcinogenesis. These results suggest that activation of SSAT by aspirin and different NSAIDs may be a common property of NSAIDs that plays an important role in their chemopreventive actions in colorectal cancer.

An activated Ki-ras was expressed in the human colon adenocarcinoma cell line Caco-2 to study the effects of Ki-ras oncogene on polyamine metabolism during gastrointestinal tumorigenesis. Multiple clones selected for expression of the mutant Ki-ras transgene displayed a suppression of transcription of a key catabolic enzyme in polyamine catabolism spermidine/spermine N1-acetyltransferase (SSAT). Gene expression analysis, with cDNA microarrays, showed that Ki-ras transfected clones had decreased levels of expression, compared to mock transfected cells, of peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor family and an important regulator of cell proliferation and differentiation. The activated Ki-ras suppressed SSAT expression by a mechanism involving the PPARgamma response element (PPRE) located at +48 bp relative to the transcription start site of the SSAT gene. Transient expression of the PPARgamma protein in Ki-ras expressing Caco-2 clones, or treatment with the PPARgamma ligand ciglitazone, led to an increase in the SSAT promoter activity. A MEK1/2 inhibitor PD98059 induced transcription of both PPARgamma and SSAT genes in the activated Ki-ras clones, suggesting that the mitogen-activated protein kinases (MAPKs) were involved in the regulation of SSAT expression by PPARgamma. We concluded that mutated Ki-ras suppressed SSAT via a transcriptional mechanism involving the PPARgamma signaling pathway., (Copyright 2004 Wiley-Liss, Inc.)

Sulindac, a non-steroidal anti-inflammatory prodrug, is metabolized into pharmacologically active sulfide and sulfone derivatives. Sulindac sulfide, but not sulindac sulfone, inhibits cyclooxygenase (COX) enzyme activities, yet both derivatives have growth inhibitory effects on colon cancer cells. Microarray analysis was used to detect COX-independent effects of sulindac on gene expression in human colorectal cells. Spermidine/sperm-ine N1-acetyltransferase (SSAT) gene, which encodes a polyamine catabolic enzyme, was induced by clinically relevant sulindac sulfone concentrations. Northern blots confirmed increased SSAT RNA levels in these colon cancer cells. Deletion analysis and mutational studies were done to map the sulindac sulfone-dependent response sequences in the SSAT 5'-flanking sequences. This led us to the identification of two peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) in the SSAT gene. PPRE-2, at +48 bases relative to the transcription start site, is required for the induction of SSAT by sulindac sulfone and is specifically bound by PPAR gamma in the Caco-2 cells as shown by transfection and gel shift experiments. PPRE-1, at-323 bases relative to the start site, is not required for the induction of SSAT by sulindac sulfone but can be bound by both PPAR delta and PPAR gamma. Sulindac sulfone reduced cellular polyamine contents in the absence but not in the presence of verapamil, an inhibitor of the export of monoacetyl diamines, inhibited cell proliferation and induced apoptosis. The induced apoptosis could be partially rescued by exogenous putrescine. These data suggest that apoptosis induced by sulindac sulfone is mediated, in part, by the COX-independent, PPAR-dependent transcriptional activation of SSAT, leading to reduced tissue polyamine contents in human colon cancer cells.

Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene (APC) and show defects in APC-dependent signaling. APC influences the expression of several genes, including the c-myc oncogene and its antagonist Mad1. Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c-myc and a modifier of APC-dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human ODC gene, which lies between two myc-binding domains. This region is known to affect ODC transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor ODC A-allele who reported using aspirin are approximately 0.10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele. Mad1 selectively suppressed the activity of the ODC promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (>or=10 microM) did not affect ODC allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the ODC polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the ODC polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention.