Bioactivity of natural biflavonoids in metabolism-related disease and cancer therapies.

Natural biflavonoids, such as amentoflavone, bilobetin, ginkgetin, isoginkgetin, taiwaniaflavone, morelloflavone, delicaflavone, hinokiflavone, and other derivatives (~ 40 biflavonoids), are isolated from Selaginella sp., Ginkgo biloba, Garcinia sp., and several other species of plants. They are able to exert therapeutic benefits by regulating several proteins/enzymes (PPAR-γ, CCAAT/enhancer-binding protein α [C/EBPα], STAT5, pancreatic lipase, PTP1B, fatty acid synthase, α-glucosidase [AG]) and insulin signaling pathways (via PI3K-AKT), which are linked to metabolism, cell growth, and cell survival mechanisms. Deregulated insulin signaling can cause complications of obesity and diabetes, which can lead to cognitive disorders such as Alzheimer's, Parkinson's, and dementia; therefore, the therapeutic benefits of these biflavones in these areas are highlighted. Since biflavonoids have shown potential to regulate metabolism, growth- and survival-related protein/enzymes, their relation to tumor growth and metastasis of cancer associated with angiogenesis are highlighted. The translational role of biflavones in cancer with respect to the inhibition of metabolism-related processes/pathways, enzymes, or proteins, such as STAT3/SHP-1/PTEN, kinesins, tissue kallikreins, aromatase, estrogen, protein modifiers, antioxidant, autophagy, and apoptosis induction mechanisms, are discussed. Finally, considering their observed bioactivity potential, oral bioavailability studies of biflavones and related clinical trials are outlined.

Analytical Method Lifecycle Management in Pharmaceutical Industry: a Review.

The adoption of Quality by Design (QbD) and Analytical Method Lifecycle Management (AMLM) concepts to ensure the quality of pharmaceutical products has been applied and proposed over the last few years. These concepts are based on knowledge gained from the application of scientific and quality risk management approaches, throughout method lifecycle to assure continuous improvement and high reliability of analytical results. The overall AMLM starts with the definition of the method's intended use through the Analytical Target Profile definition, including three stages: (1) Method Design, taking advantage of the well-known concept of QbD; (2) Method Performance Qualification; (3) Continued Method Performance Verification. This is intended to holistically align method variability with product requirements, increasing confidence in the data generated, a regulatory requirement that the pharmaceutical industry must follow. This approach views all method-related activities, such as development, validation, transfer, and routine use as a continuum and interrelated process, where knowledge and risk management are the key enablers. An increase in method robustness, cost reduction, and decreased risk failures are some of the intrinsic benefits from this lifecycle management. This approach is clearly acknowledged both by regulators and industry. The roadmap of the regulatory and industry events that mark the evolution of these concepts helps to capture the current and future expectation of the pharmaceutical framework.

Natural and Synthetic Flavonoids: Structure-Activity Relationship and Chemotherapeutic Potential for the Treatment of Leukemia.

Flavonoids and their derivatives are polyphenolic secondary metabolites with an extensive spectrum of pharmacological activities, including antioxidants, antitumor, anti-inflammatory, and antiviral activities. These flavonoids can also act as chemopreventive agents by their interaction with different proteins and can play a vital role in chemotherapy, suggesting a positive correlation between a lower risk of cancer and a flavonoid-rich diet. These agents interfere with the main hallmarks of cancer by various individual mechanisms, such as inhibition of cell growth and proliferation by arresting the cell cycle, induction of apoptosis and differentiation, or a combination of these mechanisms. This review is an effort to highlight the therapeutic potential of natural and synthetic flavonoids as anticancer agents in leukemia treatment with respect to the structure-activity relationship (SAR) and their molecular mechanisms. Induction of cell death mechanisms, production of reactive oxygen species, and drug resistance mechanisms, including p-glycoprotein efflux, are among the best-described effects triggered by the flavonoid polyphenol family.

Untangling interactions of a zinc(II) complex containing a coumarin-porphyrin unit with alkaloids in water solutions: a photophysical study.

A metal complex 1 derivative from a coumarin bearing a porphyrin unit was spectroscopically characterized and its sensing ability towards the alkaloids caffeine 2, nicotine 3 and cotinine 4 was evaluated in these studies. This probe shows to be sensitive to the alkaloids studied, where a detectable amount of 2.5 ± 0.3 µM of cotinine was determined in dam water from the Vigia Dam located in the Montoito village region, Alentejo district, Portugal. The interaction of 1 with cotinine was also verified by MALDI-TOF-MS, where it was found with peaks at 877.2 and 1053.3 m/z corresponding to the species [1H](+) and [1CotinineH](+), respectively.

Synthesis of new chlorin†e6 trimethyl and protoporphyrin†IX dimethyl ester derivatives and their photophysical and electrochemical characterizations.

In view of increasing demands for efficient photosensitizers for photodynamic therapy (PDT), we herein report the synthesis and photophysical characterizations of new chlorin e6 trimethyl ester and protoporphyrin IX dimethyl ester dyads as free bases and Zn(II) complexes. The synthesis of these molecules linked at the ß-pyrrolic positions to pyrano[3,2-c]coumarin, pyrano[3,2-c]quinolinone, and pyrano[3,2-c]naphthoquinone moieties was performed by using the domino Knoevenagel hetero Diels-Alder reaction. The α-methylenechromanes, α-methylenequinoline, and ortho-quinone methides were generated in situ from a Knoevenagel reaction of 4-hydroxycoumarin, 4-hydroxy-6-methylcoumarin, 4-hydroxy-N-methylquinolinone, and 2-hydroxy-1,4-naphthoquinone, respectively, with paraformaldehyde in dioxane. All the dyads as free bases and as Zn(II) complexes were obtained in high yields. All new compounds were fully characterized by 1D and 2D NMR techniques, UV/Vis spectroscopy, and HRMS. Their photophysical properties were evaluated by measuring the fluorescence quantum yield, the singlet oxygen quantum yield by luminescence detection, and also the triplet lifetimes were correlated by flash photolysis and intersystem crossing (ISC) rates. The fluorescence lifetimes were measured by a time-correlated single photon count (TCSPC) method, fluorescence decay associated spectra (FDAS), and anisotropy measurements. Magnetic circular dichroism (MCD) and circular dichroism (CD) spectra were recorded for one Zn(II) complex in order to obtain information, respectively, on the electronic and conformational states, and interpretation of these spectra was enhanced by molecular orbital (MO) calculations. Electrochemical studies of the Zn(II) complexes were also carried out to gain insights into their behavior for such applications.

Photodynamic inactivation of bioluminescent Escherichia coli by neutral and cationic pyrrolidine-fused chlorins and isobacteriochlorins.

Photodynamic inactivation of bioluminescent Escherichia coli in the presence of cationic chlorin and isobacteriochlorin photosensitizers (PSs) obtained from 5,10,15,20-tetrakis(pentafluorophenyl)-porphyrin is described. The spectroscopic data for the neutral and cationic derivatives and their photophysical characterizations, especially fluorescence and singlet oxygen generation capacity are also reported. The results show that there is a direct relation between the inactivation efficiency and the increasing number of charges on the molecules. The combined effect of higher wavelength absorption and number of positive charges on the PS shows a 6.1 log reduction during the inactivation process. Overall this study shows that the cationic isobacteriochlorin has high potential to be used as PS for the inactivation of Gram (-) bacteria.

Membrane-Associated Ubiquitin Ligase RING Finger Protein 152 Orchestrates Melanogenesis via Tyrosinase Ubiquitination.

Lysosomal degradation of tyrosinase, a pivotal enzyme in melanin synthesis, negatively impacts melanogenesis in melanocytes. Nevertheless, the precise molecular mechanisms by which lysosomes target tyrosinase have remained elusive. Here, we identify RING (Really Interesting New Gene) finger protein 152 (RNF152) as a membrane-associated ubiquitin ligase specifically targeting tyrosinase for the first time, utilizing AlphaScreen technology. We observed that modulating RNF152 levels in B16 cells, either via overexpression or siRNA knockdown, resulted in decreased or increased levels of both tyrosinase and melanin, respectively. Notably, RNF152 and tyrosinase co-localized at the trans-Golgi network (TGN). However, upon treatment with lysosomal inhibitors, both proteins appeared in the lysosomes, indicating that tyrosinase undergoes RNF152-mediated lysosomal degradation. Through ubiquitination assays, we found the indispensable roles of both the RING and transmembrane (TM) domains of RNF152 in facilitating tyrosinase ubiquitination. In summary, our findings underscore RNF152 as a tyrosinase-specific ubiquitin ligase essential for regulating melanogenesis in melanocytes.

Structural analysis of 2-arylidene-1-indanone derivatives by electrospray ionization tandem mass spectrometry.

RATIONALE: 2-arylidene-4-methoxy (or hydroxy)-7-methyl-1-indanone derivatives inspired from donepezil, the current drug used for the treatment of Alzheimer's disease as inhibitor of acetylcholinesterase (AChE), were studied for the first time by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). Structurally, these arylidene-indanone compounds are considered as cyclic analogues of chalcones. METHODS: ESI-MS and tandem mass spectra were acquired using a Q-TOF 2 instrument. Fragmentation patterns were analyzed by CID-MS(2-3) spectra acquired in a Q-TOF and in LXQ linear ion trap mass spectrometers using standard isolation and excitation procedures. RESULTS: All the 2-arylidene indanones have shown a common fragmentation pathway leading to a (2(1), 1')A(+) product ion at m/z 187 and the retro-aldol product ion [(2, 2(1))B(+)] that allow to establish the substitution in the B ring. The effect of electron-donating and -withdrawing substituents on these fragmentation pathways was noticed. The presence of the OCH3, OH, NO2 and Br substituents gave typical fragmentation processes that allowed their unequivocal fingerprinting. The combined loss of the ortho substituent in the B-ring plus hydrogen (H, OCH3, Br and F) is proposed to form a stable cyclic ring product. CONCLUSIONS: Arylidene indanones with different substituents on the B ring are associated with a specific fragmentation pattern. In addition, differentiation between isomers with substituents in B ring at ortho and para positions were achieved using ESI-MS/MS. These fragmentation pathways can be used to further identify and determine the fate of these molecules in all stages of drug discovery.

Electrospray tandem mass spectrometry analysis of methylenedioxy chalcones, flavanones and flavones.

RATIONALE: Several methylenedioxy chalcones, flavanones and flavones substituted with mono-, di- and trimethoxy groups have been used in the treatment of proliferative conditions like cancer and inflammatory diseases. The application of these flavonoids in biology requires an analytical method to ensure a detailed knowledge of their structures after drug metabolism. METHODS: Electrospray ionization mass (ESI-MS) and tandem mass (ESI-MS/MS) spectra were acquired using a Q-TOF 2 instrument. Fragmentation patterns and their pathways were analyzed by CID-MS(2-3) spectra acquired in a LXQ linear ion trap mass spectrometer using standard isolation and excitation procedures (activation q value of 0.25, activation time of 30 ms). ESI-MS and ESI-MS(n) conditions: spray voltage 5 kV, nitrogen 8.00 sheath gas flow rate (arb), heated capillary temperature 275°C, capillary voltage 10.99 V; tube lens voltage 75.01 V. RESULTS: The ESI-MS/MS spectra of chalcones were nearly identical to their corresponding isomeric flavanones with (0,α)A(+)/(1,3)A(+) and (0,1')B(+)/(1,4)B(+) cleavages. Other common losses are of (•)CH3, H2O, HCHO and C2H2O. The characteristic loss of C2H2O and absence of a (0,α)B(+)/(1,3)B(+) product ion allows to distinguish between the 2- or 4-methoxy-substituted chalcones and flavanones. Common losses of (•)CH3, (•)CH3 and (•)H, and C2H2O2 characteristic for the presence of methylenedioxy groups were observed in flavones. CONCLUSIONS: The substitution pattern on the B-ring leads to distinct base peak formation in the flavones. In addition, differentiation of isomers with methoxy substituents in ortho and para positions of the B-ring was achieved using MS/MS in chalcones and flavanones. This method will be helpful for identification of these compounds in biological mixtures.

Supervisory control system for monitoring a pharmaceutical hot melt extrusion process.

Continuous pharmaceutical manufacturing processes are of increased industrial interest and require uni- and multivariate Process Analytical Technology (PAT) data from different unit operations to be aligned and explored within the Quality by Design (QbD) context. Real-time pharmaceutical process verification is accomplished by monitoring univariate (temperature, pressure, etc.) and multivariate (spectra, images, etc.) process parameters and quality attributes, to provide an accurate state estimation of the process, required for advanced control strategies. This paper describes the development and use of such tools for a continuous hot melt extrusion (HME) process, monitored with generic sensors and a near-infrared (NIR) spectrometer in real-time, using SIPAT (Siemens platform to collect, display, and extract process information) and additional components developed as needed. The IT architecture of such a monitoring procedure based on uni- and multivariate sensor systems and their integration in SIPAT is shown. SIPAT aligned spectra from the extrudate (in the die section) with univariate measurements (screw speed, barrel temperatures, material pressure, etc.). A multivariate supervisory quality control strategy was developed for the process to monitor the hot melt extrusion process on the basis of principal component analysis (PCA) of the NIR spectra. Monitoring the first principal component and the time-aligned reference feed rate enables the determination of the residence time in real-time.

Natural biflavonoids as potential therapeutic agents against microbial diseases.

Microbes broadly constitute several organisms like viruses, protozoa, bacteria, and fungi present in our biosphere. Fast-paced environmental changes have influenced contact of human populations with newly identified microbes resulting in diseases that can spread quickly. These microbes can cause infections like HIV, SARS-CoV2, malaria, nosocomial Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), or Candida infection for which there are no available vaccines/drugs or are less efficient to prevent or treat these infections. In the pursuit to find potential safe agents for therapy of microbial infections, natural biflavonoids like amentoflavone, tetrahydroamentoflavone, ginkgetin, bilobetin, morelloflavone, agathisflavone, hinokiflavone, Garcinia biflavones 1 (GB1), Garcinia biflavones 2 (GB2), robustaflavone, strychnobiflavone, ochnaflavone, dulcisbiflavonoid C, tetramethoxy-6,6″-bigenkwanin and other derivatives isolated from several species of plants can provide effective starting points and become a source of future drugs. These biflavonoids show activity against influenza, severe acute respiratory syndrome (SARS), dengue, HIV-AIDS, coxsackieviral, hepatitis, HSV, Epstein-Barr virus (EBV), protozoal (Leishmaniasis, Malaria) infections, bacterial and fungal infections. Some of the biflavonoids can provide antiviral and protozoal activity by inhibition of neuraminidase, chymotrypsin-like protease, DV-NS5 RNA dependant RNA polymerase, reverse transcriptase (RT), fatty acid synthase, DNA polymerase, UL54 gene expression, Epstein-Barr virus early antigen activation, recombinant cysteine protease type 2.8 (r-CPB2.8), Plasmodium falciparum enoyl-acyl carrier protein (ACP) reductase or cause depolarization of parasitic mitochondrial membranes. They may also provide anti-inflammatory therapeutic activity against the infection-induced cytokine storm. Considering the varied bioactivity of these biflavonoids against these organisms, their structure-activity relationships are derived and wherever possible compared with monoflavones. Overall, this review aims to highlight these natural biflavonoids and briefly discuss their sources, reported mechanism of action, pharmacological uses, and comment on resistance mechanism, flavopiridol repurposing and the bioavailability aspects to provide a starting point for anti-microbial research in this area.

Nitrogen-Based Heterocyclic Compounds: A Promising Class of Antiviral Agents against Chikungunya Virus.

Arboviruses, in general, are a global threat due to their morbidity and mortality, which results in an important social and economic impact. Chikungunya virus (CHIKV), one of the most relevant arbovirus currently known, is a re-emergent virus that causes a disease named chikungunya fever, characterized by a severe arthralgia (joint pains) that can persist for several months or years in some individuals. Until now, no vaccine or specific antiviral drug is commercially available. Nitrogen heterocyclic scaffolds are found in medications, such as aristeromycin, favipiravir, fluorouracil, 6-azauridine, thioguanine, pyrimethamine, among others. New families of natural and synthetic nitrogen analogous compounds are reported to have significant anti-CHIKV effects. In the present work, we focus on these nitrogen-based heterocyclic compounds as an important class with CHIKV antiviral activity. We summarize the present understanding on this class of compounds against CHIKV and also present their possible mechanism of action.

Natural dimers of coumarin, chalcones, and resveratrol and the link between structure and pharmacology.

Natural products like coumarins, chalcones, and resveratrol have inherent biological activity in several models of diseases; therefore, their natural dimeric forms are highlighted in this review and their key structural similarities, isolation and pharmacological significance is discussed. These natural products may be dimerized during their biosynthesis, which proceeds through atom- and energy-sufficient methods involving dimeric enzymes, to provide complex structures from simple compounds. Coumarin-derived dimers features the C-C or C-O-C biaryl, terpene sidechain linkages or by cyclobutane ring and acts as inhibitors of α-glucosidase, and cytochrome p450 while some show anti-inflammatory and anti-viral activities, while chalcone-derived dimers have the 1,3-dihydroxy phenyl (resorcinol) substitution on the periphery of cyclobutane or cyclohexane ring and inhibit topoisomerase, protein tyrosine phosphatase 1B (PTP1B), and cathepsins and others possess anti-cancer, anti-inflammatory, and anti-plasmodial activities. Resveratrol-derived dimers have the resorcinol structure and are formed by oxidative coupling showing antioxidant, neuroprotective, anti-HIV, anti-tyrosinase, and cytotoxic activity. Bioavailability evidence of closely related structural monomers could be applicable to their dimeric forms. Application of bioisosteric principles to such dimeric compounds is also discussed. Overall, these dimeric natural products can provide potent templates for the natural product-based drug discovery against several diseases.

Improving reliability of Raman spectroscopy for mAb production by upstream processes during bioprocess development stages.

The use of Raman in Bioprocess development have shown great potential for process understanding and monitoring although there are still some challenges and limitations in performance when conditions such as clone, media or scale are changed during bioprocess development. This study proposes different strategies to balance the different information content of multiple mammalian cell cultivations produced during a bioprocess development program, when several conditions are investigated. The result is a pragmatic approach to PAT monitoring that serves both development and manufacturing stages. Combining risk-assessment techniques with two ways of developing monitoring calibrations (local or general models), we were able to obtain good predictive power from Raman spectroscopy used as PAT tool, when multiple cultivation conditions vary. As an example, the effects of process scale, base powder media and cell-line on Raman spectra are discussed and how using local models specific to some of these cultivation conditions, has a positive impact on calibration performance. It is shown how more accurate calibrations can be obtained using Clone-based local models, which requires less batches than usual approaches (up to 3-9). This study uses thirty-five cultivations of four different types of CHO cell lines, eight different clones, and four different scales - 2 L, 7 L, 15 L and 10,000 L - in two Cultivation Modes - fed-batch and perfusion. The aim is to serve as blueprint to how can PAT approaches be best developed in parallel to bioprocess development.

Applying near-infrared spectroscopy in downstream processing: one calibration for multiple clarification processes of fermentation media.

The use of near-infrared spectroscopy (NIRS) is demonstrated in the first downstream processing (DSP) steps of an active pharmaceutical ingredient (API) manufacturing process. The first method developed was designed to assess the API content in the filtrate stream (aqueous) of a rotary drum vacuum filter. The PLS method, built after spectral preprocessing and variable selection, had an accuracy of 0.01% (w/w) for an API operational range between 0.20 and 0.45% (w/w). The robustness and extrapolation ability of the calibration was proved when samples from ultrafiltration and nanofiltration processes, ranging from 0 to 2% (w/w), were linearly predicted (R2=0.99). The development of a robust calibration model is generally a very time-consuming task, and once established it is imperative that it can be useful for a long period of time. This work demonstrates that NIR procedures, when carefully developed, can be used in different process conditions and even in different process steps of similar unit operations.

Near-infrared reflectance spectroscopy as a process analytical technology tool in Ginkgo biloba extract qualification.

Here, we describe the use of near-infrared diffuse reflectance spectroscopy for qualification of Ginkgo biloba extract as raw material for use in pharmaceutical products. G. biloba extract shows unpredicted and uncontrolled variability in some of its quality specifications, intrinsic to its natural origin, which have influence on the manufacturing process of solid dosage forms (viz. granulation and compression). Some of these properties could not be determined by conventional quality control tests, so we investigated the use of NIR to qualify the batches of Ginkgo extract accordingly to its different features and establish a relationship with some of the manufacturing steps behaviour based on their qualification. Several approaches were evaluated, and the NIR method developed demonstrated to be sensitive to changes in important quality specifications and therefore adequate to qualify incoming batches of G. biloba extract. This could be considered a process analytical technology (PAT) application since it: (1) establishes the source of variability in a qualitative way, (2) explains its propagation to the final product quality attributes and (3) lays the basis for a control strategy to be applied in the manufacturing process.

Monitoring mAb cultivations with in-situ raman spectroscopy: The influence of spectral selectivity on calibration models and industrial use as reliable PAT tool.

Raman spectroscopy is a suitable monitoring technique for CHO cultivations. However, a thorough discussion of peaks, bands, and region assignments to key metabolites and culture attributes, and the interpretability of produced calibrations is scarce. That understanding is vital for the long-term predictive ability of monitoring models, and to facilitate lifecycle management that comply with regulatory guidelines. Several fed-batch lab-scale mAb mammalian cultivations were carried out, with in situ Raman spectroscopy used for process state estimation and attribute monitoring. The goal was to evaluate its use as a process analytical technology (PAT) tool to detect residual glucose and lactate levels, understand their dynamics and interconversion, and eventually estimate key performance culture and product quality attributes. Glucose and lactate models were optimized up to 0.31 g L-1 with 3 Latent Variables (LVs) and 0.19 g L-1 (2 LVs) accuracy, respectively. Glutamine and product titer models, were not specific and accurate enough, even though indirect calibrations were obtained with a RMSEP of 0.12 g L-1 (4 LVs) and 0.29 g L-1 (5 LVs), respectively. A critical discussion and details about the extensive work done in calibration development and optimization are provided. Namely, considering a risk-based selection of variability sources impacting sample spectra, executing designed experiments with spiked cultivations, and using advanced chemometric procedures for variable selection and model cross validation. A strategy is presented to evaluation Raman spectroscopy as a reliable PAT technology fit-for industrial use. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:659-670, 2018.

A QRM Discussion of Microbial Contamination of Non-sterile Drug Products, Using FDA and EMA Warning Letters Recorded between 2008 and 2016.

Microbial quality control of non-sterile drug products has been a concern to regulatory agencies and the pharmaceutical industry since the 1960s. Despite being an old challenge to companies, microbial contamination still affects a high number of manufacturers of non-sterile products. Consequences go well beyond the obvious direct costs related to batch rejections or product recalls, as human lives and a company's reputation are significantly impacted if such events occur. To better manage risk and establish effective mitigation strategies, it is necessary to understand the microbial hazards involved in non-sterile drug products manufacturing, be able to evaluate their potential impact on final product quality, and apply mitigation actions. Herein we discuss the most likely root causes involved in microbial contaminations referenced in warning letters issued by US health authorities and non-compliance reports issued by European health authorities over a period of several years. The quality risk management tools proposed were applied to the data gathered from those databases, and a generic risk ranking was provided based on a panel of non-sterile drug product manufacturers that was assembled and given the opportunity to perform the risk assessments. That panel identified gaps and defined potential mitigation actions, based on their own experience of potential risks expected for their processes. Major findings clearly indicate that the manufacturers affected by the warning letters should focus their attention on process improvements and microbial control strategies, especially those related to microbial analysis and raw material quality control. Additionally, the WLs considered frequently referred to failures in quality-related issues, which indicates that the quality commitment should be reinforced at most companies to avoid microbiological contaminations.LAY ABSTRACT: Microbial contamination of drug products affects the quality of non-sterile drug products produced by numerous manufacturers, representing a major risk to patients. It is necessary to understand the microbial hazards involved in the manufacturing process and evaluate their impact on final product quality so that effective prevention strategies can be implemented. A risk-based classification of most likely root causes for microbial contamination found in the warning letters issued by the US Food and Drug Administration and the European Medicines Agency is proposed. To validate the likely root causes extracted from the warning letters, a subject matter expert panel made of several manufacturers was formed and consulted. A quality risk management approach to assess microbiological contamination of non-sterile drug products is proposed for the identification of microbial hazards involved in the manufacturing process. To enable ranking of microbial contamination risks, quality risk management metrics related to criticality and overall risk were applied. The results showed that manufacturers of non-sterile drug products should improve their microbial control strategy, with special attention to quality controls of raw materials, primary containers, and closures. Besides that, they should invest in a more robust quality system and culture. As a start, manufacturers may consider investigating their specific microbiological risks, adressing their sites' own microbial ecology, type of manufacturing processes, and dosage form characteristics, as these may lead to increased contamination risks. Authorities should allow and enforce innovative, more comprehensive, and more effective approaches to in-process contamination monitoring and controls.

Improving cluster-based missing value estimation of DNA microarray data.

We present a modification of the weighted K-nearest neighbours imputation method (KNNimpute) for missing values (MVs) estimation in microarray data based on the reuse of estimated data. The method was called iterative KNN imputation (IKNNimpute) as the estimation is performed iteratively using the recently estimated values. The estimation efficiency of IKNNimpute was assessed under different conditions (data type, fraction and structure of missing data) by the normalized root mean squared error (NRMSE) and the correlation coefficients between estimated and true values, and compared with that of other cluster-based estimation methods (KNNimpute and sequential KNN). We further investigated the influence of imputation on the detection of differentially expressed genes using SAM by examining the differentially expressed genes that are lost after MV estimation. The performance measures give consistent results, indicating that the iterative procedure of IKNNimpute can enhance the prediction ability of cluster-based methods in the presence of high missing rates, in non-time series experiments and in data sets comprising both time series and non-time series data, because the information of the genes having MVs is used more efficiently and the iterative procedure allows refining the MV estimates. More importantly, IKNN has a smaller detrimental effect on the detection of differentially expressed genes.

Long Chain Alkyl Esters of Hydroxycinnamic Acids as Promising Anticancer Agents: Selective Induction of Apoptosis in Cancer Cells.

Cancer is the major cause of morbidity and mortality worldwide. Hydroxycinnamic acids (HCAs) are naturally occurring compounds and their alkyl esters may possess enhanced biological activities. We evaluated C4, C14, C16, and C18 alkyl esters of p-coumaric, ferulic, sinapic, and caffeic acids (19 compounds) for their cytotoxic activity against four human cancer cells and also examined their effect on cell cycle alteration and apoptosis induction. The tetradecyl (1c) and hexadecyl (1d) esters of p-coumaric acid and tetradecyl ester of caffeic acid (4c), but not the parental HCAs, were selectively effective against MOLT-4 (human lymphoblastic leukemia) cells with IC50 values of 0.123 ± 0.012, 0.301 ± 0.069 and 1.0 ± 0.1 µM, respectively. Compounds 1c, 1d, and 4c significantly increased apoptotic cells in sub-G1 phase and activated the caspase-3 enzyme in MOLT-4 cells. Compound 1c was 15.4 and 23.6 times more potent than doxorubicin and cisplatin, respectively, against the drug resistant MES-SA-DX5 uterine sarcoma cells. These p-coumarate esters were several times less effective against NIH/3T3 fibroblast cells. Docking studies showed that 1c may cause cytotoxicity by interaction with carbonic anhydrase IX. In conclusion, long chain alkyl esters of p-coumaric acid are promising scaffolds for selective apoptosis induction in cancer cells.