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Supplementary Figure 2 from Expression and Functions of Transmembrane Mucin MUC13 in Ovarian Cancer

Supplementary Figure Legends 1-4 from Expression and Functions of Transmembrane Mucin MUC13 in Ovarian Cancer

MUC13, a transmembrane mucin, is normally expressed in gastrointestinal and airway epithelium. Its aberrant expression has been correlated with gastric colon and cancer. However, the expression and functions of MUC13 in ovarian cancer are unknown. In the present study, the expression profile and functions of MUC13 were analyzed to elucidate its potential role in ovarian cancer diagnosis and pathogenesis. A recently generated monoclonal antibody (clone PPZ0020) was used to determine the expression profile of MUC13 by immunohistochemistry using ovarian cancer tissue microarrays and 56 additional epithelial ovarian cancer (EOC) samples. The expression of MUC13 was significantly (P < 0.005) higher in cancer samples compared with the normal ovary/benign tissues. Among all ovarian cancer types, MUC13 expression was specifically present in EOC. For the functional analyses, a full-length MUC13 gene cloned in pcDNA3.1 was expressed in a MUC13 null ovarian cancer cell line, SKOV-3. Here, we show that the exogenous MUC13 expression induced morphologic changes, including scattering of cells. These changes were abrogated through c-Jun NH2 kinase (JNK) chemical inhibitor (SP600125) or JNK2 siRNA. Additionally, a marked reduction in cell-cell adhesion and significant (P < 0.05) increases in cell motility, proliferation, and tumorigenesis in a xenograft mouse model system were observed upon exogenous MUC13 expression. These cellular characteristics were correlated with up-regulation of HER2, p21-activated kinase 1, and p38 protein expression. Our findings show the aberrant expression of MUC13 in ovarian cancer and that its expression alters the cellular characteristics of SKOV-3 cells. This implies a significant role of MUC13 in ovarian cancer. [Cancer Res 2009;69(3):765–74]

Supplementary Figure 1 from Expression and Functions of Transmembrane Mucin MUC13 in Ovarian Cancer

Integration of retroviral DNA into the host genome involves formation of integrase (IN)-DNA complexes termed intasomes. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate. The STC structure had an overall resolution of 3.36 Å and 3 Å in the conserved intasome core (CIC) region. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the CIC, similar to previously shown with the RSV octameric cleaved synaptic complex (CSC) intasome produced with IN and viral DNA only. An extensive analysis of higher-resolution STC structure helped in identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246 and S124 in CSC and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant rMUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

This study empirically investigates the impact of energy consumption (electricity consumption) and economic growth (in terms of real Gross Domestic Product) on the environmental degradation in form of CO2 emissions. The study aims to identify the interrelationship among the three variables viz. real GDP, electricity consumption & CO2 emissions. The analysis is based on the time series annual data for the period of 1971-2017. We tested the stationarity for the variables by applying Dicky Fuller test and examined the short run & long run causal relationships among electricity consumption, real GDP and CO2 emissions using Johansen Cointegration & Granger Causality approach. The Johansen cointegration test ascertains that some combinations of the two variables are cointegrated which concludes the long-term relationship among the defined variables. The results also indicate for a short run causality from electricity consumption to economic growth and to the CO2 emissions. The results conclude that India should take stringent measures to curb the surging emissions of greenhouse gases in which CO2 has a major portion.

Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes., Pandey, Bera, Shi et al. report the cryo-electron microscopy structure of the Rous sarcoma virus octameric intasome complex stabilized by a HIV-1 integrase strand transfer inhibitor. This new structure highlights the intrinsic flexibility of the distal integrase subunits and suggests that ordered conformational transitions occur within the conserved intasome core during the assembly process.

The retrovirus integrase (IN) inserts the viral cDNA into the host DNA genome. Atomic structures of five different retrovirus INs complexed with their respective viral DNA or branched viral/target DNA substrates have indicated these intasomes are composed of IN subunits ranging from tetramers, to octamers, or to hexadecamers. IN precursors are monomers, dimers, or tetramers in solution. But how intasome assembly is controlled remains unclear. Therefore, we sought to unravel the functional mechanisms in different intasomes. We produced kinetically stabilized Rous sarcoma virus (RSV) intasomes with human immunodeficiency virus type 1 strand transfer inhibitors that interact simultaneously with IN and viral DNA within intasomes. We examined the ability of RSV IN dimers to assemble two viral DNA molecules into intasomes containing IN tetramers in contrast to one possessing IN octamers. We observed that the last 18 residues of the C terminus (“tail” region) of IN (residues 1–286) determined whether an IN tetramer or octamer assembled with viral DNA. A series of truncations of the tail region indicated that these 18 residues are critical for the assembly of an intasome containing IN octamers but not for an intasome containing IN tetramers. The C-terminally truncated IN (residues 1–269) produced an intasome that contained tetramers but failed to produce an intasome with octamers. Both intasomes have similar catalytic activities. The results suggest a high degree of plasticity for functional multimerization and reveal a critical role of the C-terminal tail region of IN in higher order oligomerization of intasomes, potentially informing future strategies to prevent retroviral integration.

The purpose of this chapter is to identify the dimensions of green supply chain and their impact on manufacturing practices. In this study, the authors used two extended strategies. First thorough review of literature was done considering articles from reputed journals. Second the factors identified from literature review was further refined through experts by forming a problem solving group consisting of seven experts from the manufacturing sector. These factors were used to develop the green supply chain management model using Interpretive structural modeling. Further MICMAC analysis was used to identify the driving and dependence power of the factors. The results of the analysis are very encouraging. Finally, the authors have presented the relationship management strategy for sustainable manufacturing practices.

Many LCA based viability studies have already been done for the production of green diesel from microalgae, still a comprehensive LCA, has not yet been undertaken. Current study aims to find out if the Net Energy Balance(NEB) can further be increased by using a combination of many available agronomical practices & the techniques of production of green diesel from microalgae. The results show that neither open raceway pond nor Photobioreactor routes (Wet and dry routes) yield positive energy balance. The production of green diesel via open raceway pond, both in dry as well as wet route, have less negative NEB and comparatively higher NER than the photobioreactors. Comparison says that open raceway pond dry route has slightly higher value for NER than the wet route. Even with the best possible route (open raceway pond dry route), the total energy use is almost 5 times more than the energy produced, with a negative NEB of 4.07 MJ & very low NER value of 0.20. Study concludes that R & D in the area of green diesel production from microalgae has yet to go a long way & has a huge scope to further lower its input energy demand for biofuel production.

Electricity theft is a growing problem worldwide. India, like many other developing countries, is in the grips of electricity theft. To address this challenge, the country introduced various technological solutions, which failed to realize their promised potential. Despite little success, majority of theft-combat interventions continue to emphasize technology and downplay psycho-social factors. The latter calls for deep exploration as power-utility employees have been found to connive with consumers in majority of electricity thefts. Though, employee theft has been explained by distinct perspectives like sociology, psychology, criminology, organizational science etc., none describes the phenomenon completely. In order to gain a holistic understanding, it is imperative to integrate the said perspectives. That's precisely what this paper attempts- It proposes an integrated conceptual framework of the psycho-social factors that predispose employees to collude with consumers in electricity theft.

Artificial intelligent methods are being extensively used for oil price forecasting as an alternate approach to conventional techniques. There has been a whole spectrum of artificial intelligent techniques to overcome the difficulties of complexity and irregularity in oil price series. The potential of AI as a design tool for oil price forecasting has been reviewed in this study. The following price forecasting techniques have been covered: (i) artificial neural network, (ii) support vector machine, (iii) wavelet, (iv) genetic algorithm, and (v) hybrid systems. In order to investigate the state of artificial intelligent models for oil price forecasting, thirty five research papers (published during 2001 to 2013) had been reviewed in form of table (for ease of comparison) based on the following parameters: (a) input variables, (b) input variables selection method, (c) data characteristics (d) forecasting accuracy and (e) model architecture. This review reveals procedure of AI methods used in complex oil price related studies. The review further extended above overview into discussions regarding specific shortcomings that are associated with feature selection for designing input vector, and then concluded with future insight on improving the current state-of-the-art technology.

India has tremendous potential for generating clean electricity through Renewable Energy Sources (RES) namely Hydro, Wind and Solar. This potential has been duly recognized and shows India׳s consciousness for reducing carbon footprint as a developing nation. Government of India with an aim to promote clean energy launched Jawaharlal Nehru National Solar Mission (JNNSM) on 11th January 2010, which is one of the eight missions under National Action Plan on Climate Change (NAPCC–2008). This mission visions to install 22,000 MW through grid connected and off grid power plants. Achieving an installed capacity of this quantum is a task full of challenges. To list the possible challenges and suggest a way forward, there is a need to study solar energy sector in India in the past, which has motivated the authors to discuss the evolution of solar energy in India since independence. Through this paper authors have tried to outline the journey of solar energy in India since 1950 till date and highlight the potential issues as barriers and challenges which could impact the ambitious mission taken up by Government of India. This study will help decision makers and various stakeholders to understand the current status, barriers and challenges for better planning and management in the field of solar energy.

We determined conditions to produce milligram quantities of the soluble Rous sarcoma virus (RSV) synaptic complex that is kinetically trapped by HIV strand transfer inhibitors (STIs). Concerted integration catalyzed by RSV integrase (IN) is effectively inhibited by HIV STIs. Optimized assembly of the RSV synaptic complex required IN, a gain-of-function 3'-OH-recessed U3 oligonucleotide, and an STI under specific conditions to maintain solubility of the trapped synaptic complex at 4 °C. A C-terminal truncated IN (1-269 residues) produced a homogeneous population of trapped synaptic complex that eluted at ∼ 151,000 Da upon Superdex 200 size-exclusion chromatography (SEC). Approximately 90% of input IN and DNA are incorporated into the trapped synaptic complex using either the C-terminally truncated IN or wild type IN (1-286 residues). No STI is present in the SEC running buffer suggesting the STI-trapped synaptic complex is kinetically stabilized. The yield of the trapped synaptic complex correlates with the dissociative half-life of the STI observed with HIV IN-DNA complexes. Dolutegravir, MK-2048, and MK-0536 are equally effective, whereas raltegravir is ∼ 70% as effective. Without an STI present in the assembly mixture, no trapped synaptic complex was observed. Fluorescence and mass spectroscopy analyses demonstrated that the STI remains associated with the trapped complex. SEC-multiangle light scattering analyses demonstrated that wild type IN and the C-terminal IN truncation are dimers that acted as precursors to the tetramer. The purified STI-trapped synaptic complex contained a tetramer as shown by cross-linking studies. Structural studies of this three-domain RSV IN in complex with viral DNA may be feasible.

India is one of the most rapidly developing countries in the world. It is witnessing growing industrialization and thus development. Such rapid development needs energy to progress, which further makes India an energy hungry nation. Currently India depends mainly upon fossil fuels and thus has to pay a huge bill at the end of every contractual period. These bills can be shortened and the expenditures brought down by using and exploiting non-conventional sources of energy. India holds a huge potential for such non-conventional sources of energy. The rapid development of India is not just pressing hard upon its resources but forcing expenditures on the same. There are also some neglected side effects of this development process like, generation of waste. A population of 1.2 billion is generating 0.5 kg per person every day. This, sums up to a huge pile of waste, which is mostly landfilled in the most unhygienic manner possible. Such unmanaged waste not only eats up resources but demands expenditure as well. This can lead to the downfall of an economy and degradation of the nation. Thus, the paper presents waste to energy as a solution to both the problems stated above, using which not only can we reduce the amount of waste, but also produce energy from the same, thus achieving our goal of waste management as well as energy security. The paper presents the current status, major achievements and future aspects of waste to energy in India which will help decision makers, planners and bodies involved in the management of municipal solid waste understand the current status challenges and barriers of MSWM in India for further better planning and management.

India is poised to spend over USD 5.8 billion as part of the National Smart Grid Mission aimed to alleviate India's ailing power sector as part of its 12th Five year plan (2012–2017). The federal government sponsored Restructured Accelerated Power Development and Reforms Program (R-APDRP) is also focused on building ICT capability in the state electricity boards. Presently however, there is no power sector specific cyber security mandates or policies in India. The Stuxnet, Shamoon and Anonymous incidents have shown that cyber attacks can cause significant damage and pose a risk to National Critical Infrastructure. A lack of security planning as part of designing the Smart grids can potentially leave gaping holes in the country's power sector stability. The paper highlights key cyber security threats across the entire power sector value chain—from generation, to transmission and distribution. It is aimed at building the case for power sector specific cyber security regulations based on the experience of regulators in other critical infrastructure sectors like Banking and Telecom in India and power sector regulations internationally.

Microalgae are receiving increasing attention worldwide as an alternative and renewable source for energy production. Through various conversion processes, microalgae can be used to produce many different kinds of biofuels, which include biodiesel, bio-syngas, bio-oil, bio-ethanol, and bio-hydrogen. However, large scale production of microalgal biofuels, via many available conversion techniques, faces a number of technical challenges which have made the current growth and development of the algal biofuel industry economically unviable. Therefore, in addition to algae culture and growth, it is also essential to develop cost-effective technologies for efficient biomass harvesting, lipid extraction and biofuels production. This review aims to collate and present an overview of current harvesting, oil extraction and biofuels production technologies from microalgae. Since much of the current studies on oil extraction are focused on biodiesel production from microalga, this study, apart from discussing the various biodiesel production techniques in the later sections, has also done a detailed discussion on the production techniques of other biofuels.

PurposeThe fact that complaints regarding general insurance claims are three times as numerous as those of life insurance claims suggests that claims behaviour of general insurers be investigated to minimize operating losses and ensure operational excellence. This paper seeks to address this issue.Design/methodology/approachStudy of variance and factor analysis has been undertaken to achieve the objective of identifying factors which govern claims in general insurance business. In order to understand the dependency of claims over the sectors and segments, statistical hypothesis testing along with cross tab analysis has been conducted. The study also evaluates the relationship of these factors over the sectors and segments by running a multiple regression.FindingsAn empirical result of the study proves that there exists an association between type of sectors, i.e. public and private and segments of insurance namely fire, marine and miscellaneous. The study also suggests a claim projection model for the general insurance players.Research limitations/implicationsExclusion of specialized players due to the reason being new entrants and in order to maintain common parlance of sectors may be a limitation to this study.Originality/valueThe study recommends that insurance players should not treat the claims settlement strategies in isolation of segments. The claims projection model as suggested in the study may prove to be extremely helpful in projecting the claims and in turn reduce the increasing underwriting losses.

APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.

The hunt for a compound which inhibits the HIV-1 integrase had been painstakingly difficult. Integrase is essential for viral replication as it mediates the integration of the viral DNA genome into the host DNA resulting in the establishment of the permanent provirus. Persistent efforts have resulted in the discovery of Raltegravir (Isentress, MK-0518), the first integrase inhibitor approved by US Food and Drug Administration for the treatment in HIV-1 infected patients. Numerous clinical studies with raltegravir have found it to be safe and effective in treatment naïve as well as treatment experienced patients. Adverse events associated with raltegravir based therapy are milder compared to previously available regimens. Raltegravir is metabolized primarily via glucuronidation mediated by uridine diphosphate glucuronosyltransferase and has a favorable pharmacokinetics independent of age, gender, race, food, and drug-drug interactions. Within a short period of time of its introduction, raltegravir has been included as one of DHHS recommended preferred regimen for the treatment of HIV-1 infection in treatment naïve patients.

In the current scenario of depleting energy resources, increasing food insecurity and global warming, Jatropha has emerged as a promising energy crop for India. The aim of this study is to examine the life cycle energy balance for Jatropha biodiesel production and greenhouse gas emissions from post-energy use and end combustion of biodiesel, over a period of 5 years. It’s a case specific study for a small scale, high input Jatropha biodiesel system. Most of the existing studies have considered low input Jatropha biodiesel system and have used NEB (Net energy balance i.e. difference of energy output and energy input) and NER (Net energy ratio i.e. ratio of energy output to energy input) as indicators for estimating the viability of the systems. Although, many of them have shown these indicators to be positive, yet the values are very less. The results of this study, when compared with two previous studies of Jatropha, show that the values for these indicators can be increased to a much greater extent, if we use a high input Jatropha biodiesel system. Further, when compared to a study done on palm oil and Coconut oil, it was found even if the NEB and NER of biodiesel from Jatropha were lesser in comparison to those of Palm oil and Coconut oil, yet, when energy content of the co-products were also considered, Jatropha had the highest value for both the indicators in comparison to the rest two.

The assembly mechanism for the human immunodeficiency virus type-1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO4 was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3′ OH recessed and blunt ended U5 long terminal repeat (LTR) oligonucleotides (ODN) substrates (19 bp to 42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that a IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with increasing length of the ODN, starting from the recessed 18/20 bp or 19/21 bp set to the 31/33 bp and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ~32 bp DNaseI protective footprint suggesting, viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to an 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate to attempt crystallization of an IN-DNA complex in the absence or presence of strand transfer inhibitors.

MUC13, a transmembrane mucin, is normally expressed in gastrointestinal and airway epithelium. Its aberrant expression has been correlated with gastric colon and cancer. However, the expression and functions of MUC13 in ovarian cancer are unknown. In the present study, the expression profile and functions of MUC13 were analyzed to elucidate its potential role in ovarian cancer diagnosis and pathogenesis. A recently generated monoclonal antibody (clone PPZ0020) was used to determine the expression profile of MUC13 by immunohistochemistry using ovarian cancer tissue microarrays and 56 additional epithelial ovarian cancer (EOC) samples. The expression of MUC13 was significantly (P < 0.005) higher in cancer samples compared with the normal ovary/benign tissues. Among all ovarian cancer types, MUC13 expression was specifically present in EOC. For the functional analyses, a full-length MUC13 gene cloned in pcDNA3.1 was expressed in a MUC13 null ovarian cancer cell line, SKOV-3. Here, we show that the exogenous MUC13 expression induced morphologic changes, including scattering of cells. These changes were abrogated through c-Jun NH2 kinase (JNK) chemical inhibitor (SP600125) or JNK2 siRNA. Additionally, a marked reduction in cell-cell adhesion and significant (P < 0.05) increases in cell motility, proliferation, and tumorigenesis in a xenograft mouse model system were observed upon exogenous MUC13 expression. These cellular characteristics were correlated with up-regulation of HER2, p21-activated kinase 1, and p38 protein expression. Our findings show the aberrant expression of MUC13 in ovarian cancer and that its expression alters the cellular characteristics of SKOV-3 cells. This implies a significant role of MUC13 in ovarian cancer. [Cancer Res 2009;69(3):765–74]

Human immunodefi ciency virus type-1 integrase (IN) is a new and novel target for inhibitors. Strand transfer inhibitors effectively prevent concerted integration of viral DNA by IN into the host chromosomes. Raltegravir is the fi rst approved strand transfer inhibitor for the treatment of HIV-1/AIDS. We propose a mechanistic hypothesis as to "when and where" these inhibitors are active in virus-infected cells. Using native agarose gel electrophoresis, we identified a transient synaptic complex (SC) wherein IN non-covalently juxtaposes two viral DNA ends. SC possesses many properties associated with the cytoplasmic preintegration complex (PIC) in infected cells, including concerted integration. Our results show that the strand transfer inhibitors effectively "trap" or inactivate the SC preventing concerted integration. It follows that the IN-viral DNA complex is "trapped" by the inhibitors via a transient intermediate within the cytosolic PIC before entry into the nucleus.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) inserts the viral DNA genome into host chromosomes. Here, by native agarose gel electrophoresis, using recombinant IN with a blunt-ended viral DNA substrate, we identified the synaptic complex (SC), a transient early intermediate in the integration pathway. The SC consists of two donor ends juxtaposed by IN noncovalently. The DNA ends within the SC were minimally processed (∼15%). In a time-dependent manner, the SC associated with target DNA and progressed to the strand transfer complex (STC), the nucleoprotein product of concerted integration. In the STC, the two viral DNA ends are covalently attached to target and remain associated with IN. The diketo acid inhibitors and their analogs effectively inhibit HIV-1 replication by preventing integration in vivo. Strand transfer inhibitors L-870,810, L-870,812, and L-841,411, at low nM concentrations, effectively inhibited the concerted integration of viral DNA donor in vitro. The inhibitors, in a concentration-dependent manner, bound to IN within the SC and thereby blocked the docking onto target DNA, which thus prevented the formation of the STC. Although 3′-OH recessed donor efficiently formed the STC, reactions proceeding with this substrate exhibited marked resistance to the presence of inhibitor, requiring significantly higher concentrations for effective inhibition of all strand transfer products. These results suggest that binding of inhibitor to the SC occurs prior to, during, or immediately after 3′-OH processing. It follows that the IN-viral DNA complex is “trapped” by the strand transfer inhibitors via a transient intermediate within the cytoplasmic preintegration complex.

The retrovirus integrase (IN) is responsible for integration of the reverse transcribed linear cDNA into the host DNA genome. First, IN cleaves a dinucleotide from the 3' OH blunt ends of the viral DNA exposing the highly conserved CA sequence in the recessed ends. IN utilizes the 3' OH ends to catalyze the concerted integration of the two ends into opposite strands of the cellular DNA producing 4 to 6 bp staggered insertions, depending on the retrovirus species. The staggered ends are repaired by host cell machinery that results in a permanent copy of the viral DNA in the cellular genome. Besides integration, IN performs other functions in the replication cycle of several studied retroviruses. The proper organization of IN within the viral internal core is essential for the correct maturation of the virus. IN plays a major role in reverse transcription by interacting directly with the reverse transcriptase and by binding to the viral capsid protein and a cellular protein. Recruitment of several other host proteins into the viral particle are also promoted by IN. IN assists with the nuclear transport of the preintegration complex across the nuclear membrane. With several retroviruses, IN specifically interacts with different host protein factors that guide the preintegration complex to preferentially integrate the viral genome into specific regions of the host chromosomal target. Human gene therapy using retrovirus vectors is directly affected by the interactions of IN with these host factors. Inhibitors directed against the human immunodeficiency virus (HIV) IN bind within the active site of IN containing viral DNA ends thus preventing integration and subsequent HIV/AIDS.

Geopolitical and economic events had strong impact on crude oil markets for over 40 years. Oil prices steadily rose for several years and in July 2008 stood at a record high of $145 per barrel. Further, it plunged to $43 per barrel by end of 2008. There is need to identify appropriate features (factors) explaining the characteristics of oil markets during booming and downturn period. Feature selection can help in identifying the most informative and influential input variables before and after financial crisis. The study used an extended version of MI3 algorithm i.e. I2MI2 algorithm together with general regression neural network as forecasting engine to examine the explanatory power of selected features and their contribution in driving oil prices. The study used features selected from proposed methodology for one-month ahead and twelve-month ahead forecast horizon. The forecast from the proposed methodology outperformed in comparison to EIA's STEO estimates. Results shows that reserves and speculations were main players before the crisis and the overall mechanism was broken due to 2008 global financial crisis. The contribution of emerging economy (China) emerged as important variable in explaining the directions of oil prices. EPPI and CPI remain the building blocks before and after crisis while influence of Non-OECD consumption rises after the crisis.

The disappointing current therapeutic approaches for pancreatic cancer (PC) represent an urgent need for the development of novel methods to control the disease. Based on a recent report on the effectiveness of pancreatic enzyme therapy, we examined the effect of porcine pancreatic enzyme extracts (PPE) on human PC xenografts in nude mice.The malignant human PC cell line AsPC1 was transplanted into the pancreas of male beige XID nude mice that were treated or not with PPE in drinking water. The survival, size, and volume of tumors, plasma pancreatic enzyme levels, fecal fat, and urine were examined as were the expression of transforming growth factor alpha, insulinlike growth factor-I, epidermal growth factor, epidermal growth factor receptor, apoptosis, and proliferation rate of tumor cells.PPE-treated mice survived significantly longer than the control group (P0.002). Tumors in the PPE-treated group were significantly smaller than in the control group. All mice in the control group showed steatorrhea, hyperglucosuria, hyperbilirubinuria, and ketonuria at early stages of tumor growth, whereas only a few in the treated group showed some of these abnormalities at the final stage. There were no differences in the expression of growth factors, epidermal growth factor receptor, or the apoptotic rate between the tumors of treated and control mice.The treatment with PPE significantly prolongs the survival of mice with human PC xenografts and slows the tumor growth. The data indicate that the beneficial effect of PPE on survival is primarily related to the nutritional advantage of the treated mice.

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase-DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNA remained elusive. Here we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein-DNA and protein-protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.

Human immunodeficiency virus type 1 (HIV-1) integrase inhibitors belong to a novel class of antiretroviral drugs with high potency and better tolerability. Elvitegravir (EVG) is the second integrase inhibitor approved by the US Food and Drug Administration when administered in combination with a novel pharmacoenhancer, cobicistat (COBI), and two nucleoside/nucleotide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF). This combination of drugs (EVG/COBI/FTC/TDF) developed and marketed by Gilead Sciences Inc. (Foster City, CA, USA) as STRIBILD®, is the first integrase inhibitor-based single-tablet regimen administered once-daily. In the USA, it has been approved for use in antiretroviral treatment-naive HIV-1 patients with estimated creatinine clearance of >70 mL/min. The Department of Health and Human Services has approved EVG/COBI/FTC/TDF as one of preferred first-line regimens for HIV-1 treatment. In Europe, the European Medicines Agency has approved STRIBILD in treatment-naive patients as well as in patients having no resistant mutation to any of the antiviral agents contained in STRIBILD. Its availability as a fixed-dose combination and once-daily dosage makes the adherence highly likely. However, it also discounts the possibility of dosage adjustment if needed.

Twenty-three propane- and butane-utilizing bacteria were isolated from soil samples collected from oilfields. Three of them have been identified as Rhodococcus sp. IMT35, Pseudomonas sp. IMT37 and Pseudomonas sp. MT40. SDS-PAGE analysis of the membrane of Rhodococcus sp. IMT35 revealed the presence of at least four polypeptides induced by propane. Polyclonal antibody raised against a 58 kDa polypeptide from Rhodococcus sp. IMT35 specifically detected bacteria which were actively utilizing propane or butane. Immunoscreening of a genomic library in lambdagt11 with this antibody resulted in isolation of a clone containing a 4.9 kb EcoRI genomic DNA fragment. This 4.9 kb DNA fragment was found to hybridize specifically with organisms which could grow on propane or butane. This fragment could therefore be used as a probe for detection of such bacteria. A 2.3 kb fragment having an ORF encoding a polypeptide of 54 kDa was identified by screening a genomic library of Pseudomonas sp. IMT37 with this 4.9 kb EcoRI fragment. The sequence of the ORF (designated orf54) was found to be novel. Primer extension and S1 nuclease mapping showed that transcription of the ORF starts at base 283 and it had sequences upstream similar to that of a Pseudomonas promoter (-12, -24 type). Disruption of the ORF by a kanamycin ('kan') cassette prevented the organism from growing on any alkane but did not affect its ability to utilize the respective alkanols and acids, indicating that alcohol dehydrogenase and subsequent steps in the pathway remained unaltered. The mutants had no detectable level of butane monooxygenase activity. Therefore, the product of this gene plays a crucial role in the first step of the pathway and is an essential component of monooxygenase. The findings imply that this bacterium either employs a common genetic and metabolic route or at least shares the product of this gene for utilization of many alkanes.

Integration of human immunodeficiency virus cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt ends to form the synaptic complex, which is the intermediate in the concerted integration pathway. The synaptic complex is inactivated by strand transfer inhibitors (STI) with IC50 values of ∼ 20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on a native agarose gel that was produced in the presence of > 200 nM STI, termed the IN–single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus, producing an ∼ 32-bp DNase I protective footprint. In the presence of raltegravir (RAL), MK-2048, and L-841,411, IN incorporated ∼ 20–25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤ 5% of input DNA). The formation of the ISD complex was not dependent on 3′OH processing, and the DNA was predominantly blunt ended in the complex. The RAL-resistant IN mutant N155H weakly forms the ISD complex in the presence of RAL at ∼ 25% level of wild-type IN. In contrast, MK-2048 and L-841,411 produced ∼ 3-fold to 5-fold more ISD than RAL with N155H IN, which is susceptible to these two inhibitors. The results suggest that STI are slow-binding inhibitors and that the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex.

Raltegravir is an FDA approved inhibitor directed against human immunodeficiency virus type 1 (HIV-1) integrase (IN). In this study, we investigated the mechanisms associated with multiple strand transfer inhibitors capable of inhibiting concerted integration by HIV-1 IN. The results show raltegravir, elvitegravir, MK-2048, RDS 1997, and RDS 2197 all appear to encompass a common inhibitory mechanism by modifying IN-viral DNA interactions. These structurally different inhibitors bind to and inactivate the synaptic complex, an intermediate in the concerted integration pathway in vitro. The inhibitors physically trap the synaptic complex, thereby preventing target DNA binding and thus concerted integration. The efficiency of a particular inhibitor to trap the synaptic complex observed on native agarose gels correlated with its potency for inhibiting the concerted integration reaction, defined by IC(50) values for each inhibitor. At low nanomolar concentrations (50 nM), raltegravir displayed a time-dependent inhibition of concerted integration, a property associated with slow-binding inhibitors. Studies of raltegravir-resistant IN mutants N155H and Q148H without inhibitors demonstrated that their capacity to assemble the synaptic complex and promote concerted integration was similar to their reported virus replication capacities. The concerted integration activity of Q148H showed a higher cross-resistance to raltegravir than observed with N155H, providing evidence as to why the Q148H pathway with secondary mutations is the predominant pathway upon prolonged treatment. Notably, MK-2048 is equally potent against wild-type IN and raltegravir-resistant IN mutant N155H, suggesting this inhibitor may bind similarly within their drug-binding pockets.

A macromolecular nucleoprotein complex in retrovirus-infected cells, termed the preintegration complex, is responsible for the concerted integration of linear viral DNA genome into host chromosomes. Isolation of sufficient quantities of the cytoplasmic preintegration complexes for biochemical and biophysical analyses is difficult. We investigated the architecture of human immunodeficiency virus type-1 (HIV-1) nucleoprotein complexes involved in the concerted integration pathway in vitro. HIV-1 integrase (IN) non-covalently juxtaposes two viral DNA termini forming the synaptic complex, a transient intermediate in the integration pathway and shares properties associated with the preintegration complex. IN slowly processes two nucleotides from the 3′ OH ends and performs the concerted insertion of two viral DNA ends into target DNA. IN remains associated with the concerted integration product, termed the strand transfer complex. The synaptic complex and strand transfer complex can be isolated on native agarose gels for biochemical and biophysical analyses. In this report, in-gel fluorescence resonance energy transfer measurements demonstrated that the energy transfer efficiencies between the juxtaposed Cy3 and Cy5 5′-end labeled viral DNA ends in the synaptic complex (0.68 ± 0.09) was significantly different than observed in the strand transfer complex (0.07 ± 0.02). The calculated distances were 46 ± 3 Å and 83 ± 5 Å, respectively. DNaseI footprint analysis of the complexes revealed that IN protects U5 and U3 DNA sequences up to ~32 bp from the end, suggesting two IN dimers were bound per terminus. Enhanced DNaseI cleavages were observed at nucleotide positions 6 and 9 from the terminus on U3 but not on U5 suggesting independent assembly events. Protein-protein cross-linking of IN within these complexes revealed the presence of dimers, tetramers, and a larger-size multimer (>120 Kd). Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends prior to the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex.

Retrovirus integrase (IN) integrates the viral linear DNA genome ( approximately 10 kb) into a host chromosome, a step which is essential for viral replication. Integration occurs via a nucleoprotein complex, termed the preintegration complex (PIC). This article focuses on the reconstitution of synaptic complexes from purified components whose molecular properties mirror those of the PIC, including the efficient concerted integration of two ends of linear viral DNA into target DNA. The methods described herein permit the biochemical and biophysical analyses of concerted integration. The methods enable (1) the study of interactions between purified recombinant IN and its viral DNA substrates at the molecular level; (2) the identification and characterization of nucleoprotein complexes involved in the human immunodeficiency virus type-1 (HIV-1) concerted integration pathway; (3) the determination of the multimeric state of IN within these complexes; (4) dissection of the interaction between HIV-1 IN and cellular proteins such as lens epithelium-derived growth factor (LEDGF/p75); (5) the examination of HIV-1 Class II and strand transfer inhibitor resistant IN mutants; (6) the mechanisms associated with strand transfer inhibitors directed against HIV-1 IN that have clinical relevance in the treatment of HIV-1/AIDS.

The “strand transfer inhibitors” of human immunodeficiency virus type-1 (HIV-1) integrase (IN), so named because of their pronounced selectivity for inhibiting strand transfer over 3′ OH processing, block virus replication in vivo and ex vivo and prevent concerted integration in vitro. We explored the kinetics of product formation and strand transfer inhibition within reconstituted synaptic complexes capable of concerted integration. Synaptic complexes were formed with viral DNA donors containing either two blunt ends, two 3′-OH-processed ends, or one of each. We determined that one blunt end within a synaptic complex is a sufficient condition for low-nanomolar-range strand transfer inhibition with naphthyridine carboxamide inhibitors L-870,810 and L-870,812. We further explored the catalytic properties and drug resistance profiles of a set of clinically relevant strand transfer inhibitor-resistant HIV-1 IN mutants. The diketo acids and naphthyridine carboxamides, mechanistically similar but structurally distinct strand transfer inhibitors, each select for a distinct set of drug resistance mutations ex vivo. The S153Y and N155S IN resistance mutants were selected with the diketo acid L-841,411, and the N155H mutant was selected with L-810,812. Each mutant exhibited some degree of catalytic impairment relative to the activity of wild type IN, although the N155H mutant displayed near-wild-type IN activities. The resistance profiles indicated that the S153Y mutation potentiates susceptibility to L-870,810 and L-870,812, while the N155S mutation confers resistance to L-870,810 and L-870,812. The N155H mutation confers resistance to L-870,810 and potentiates susceptibility to L-841,411. This study illuminates the interrelated mechanisms of concerted integration, strand transfer inhibition, and resistance to strand transfer inhibitors.

Human transcriptional coactivator p75/lens epithelium-derived growth factor (LEDGF) binds human immunodeficiency virus type 1 (HIV-1) integrase (IN). We studied the effects of LEDGF on the assembly and activity of HIV-1 synaptic complexes, which, upon association with a target, mediate concerted integration of viral DNA substrates in vitro. We found that while augmenting single-ended viral DNA integration into target DNA, the host factor was able to either stimulate or abrogate concerted integration in a concentration-dependent manner. LEDGF modestly stimulated (two- to threefold) concerted integration at low molar ratios to IN (1, apparently due to the disruption of IN-IN interactions essential for the formation of active synaptic complexes prior to their association with a circular target. The isolated IN binding domain of LEDGF was sufficient to stimulate and inhibit concerted integration, as observed with full-length protein, albeit at lower efficiencies. Our data show that LEDGF differentially affects IN-DNA complexes mediating single-ended viral DNA integration and synaptic complexes mediating concerted integration. Synaptic complexes associated with target, termed strand transfer complexes, are resistant to disruption by high concentrations of LEDGF. The results suggest that LEDGF may influence HIV-1 integration in vivo.