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AbstractThe impact of COVID-19 infection on individuals with small cell lung cancer (SCLC) poses a serious threat. Unfortunately, the molecular basis of this severe comorbidity has yet to be elucidated. The present study addresses this gap utilizing publicly available omics data of COVID-19 and SCLC to explore the key molecules and associated pathways involved in the convergence of these diseases. Findings revealed 402 genes, that exhibited differential expression patterns in SCLC patients and also play a pivotal role in COVID-19 pathogenesis. Subsequent functional enrichment analyses identified relevant ontologies and pathways that are significantly associated with these genes, revealing important insights into their potential biological, molecular and cellular functions. The protein-protein interaction network, constructed under four combinatorial topological assessments, highlighted SMAD3, CAV1, PIK3R1, and FN1 as the primary components to this comorbidity. Our results suggest that these components significantly regulate this cross-talk triggering the PI3K-AKT and TGF-β signaling pathways. Lastly, this study made a multi-step computational attempt and identified corylifol A and ginkgetin from natural sources that can potentially inhibit these components. Therefore, the outcomes of this study offer novel perspectives on the common molecular mechanisms underlying SCLC and COVID-19 and present future opportunities for drug development.Communicated by Ramaswamy H. Sarma

In this paper, soil water characteristic curves (SWCCs) were reconstructed through simultaneous field measurement of soil water content and suction value. The objective of this study was to evaluate whether field-based monitoring could capture the different SWCC paths in various seasons. Moreover, field measurement of SWCC makes it possible to quantify the hysteresis phenomenon, analyzes the effect of various temporal resolutions on SWCC, and highlights the differences in reconstructed SWCC for the same soil during various weather conditions. Field data from one expansive subgrade site in Texas, USA were collected through moisture and suction sensors over a 3-year monitoring period. Analyzing the rainfall and evapotranspiration data, three distinct wetting and drying cycles were identified which allowed correct discrimination of the cycles. The pattern of changes in SWCC was identified in the various wetting and drying cycles. While the field data was fitted through the Van Genuchten equation, variations of the unsaturated soil parameters (α, n, m) were identified in various cycles. Air entry value indicator parameter αranged from 0.05 to 0.11 kPa−1while the slope of the SWCC curve parameter nvalue varied from 1.1 to 1.45. Comparing these results with previous studies, it was concluded that wetting and drying cycle parameters do not have any predefined correlation. However, these unsaturated parameters required for numerical modeling are dependent on the climatic events happening year-round.

A wear test was performed under the long-used machine oil sliding environment on Cu-based alloys Al-bronze and α-brass. A pin-on-disc wear testing method was applied where normal pressure of 0.255 to 2.55 MPa and a constant sliding speed of 0.641 m/s were considered. For the assessment dry and fresh machine oil sliding environments were also conducted. The results from the experiment demonstrated that the wear rate and friction coefficient in dry sliding condition were much greater for their direct contact but lower under machine oil due to the reduced roughness by the sealing effect as oil forms a thin lubricating film between the contact surfaces. Used oil displayed some degree of higher wear rate along with friction coefficient due to heavy and harmful chemical compounds in it. Al-bronze performed the better wear properties with lower wear rate and coefficient of friction for all the environments as it achieved the strength through different intermetallic formations. In case of α-brass, it had little effect on wear characteristics. Examined by optical microscopy and SEM analysis, worn surfaces showed that Al-bronze improved wear resistance through mild and smooth abrasive grooves filled with oxides in dry sliding conditions. In case of oil sliding environment, smooth surfaces were created by the resistance of the oil film to the direct contact between the surfaces. Used oil sliding conditions reduced the surface smoothness of the Cu and Cu-based alloys for the presence of damaging chemical compounds.

This study explores hybridized machine learning (ML) techniques to predict autogenous shrinkage (AS) in ultra-high-performance concrete (UHPC). The ensemble model, namely random forest (RF), extra tree regressor (ETR), light gradient boosting machine (LGBM), and extended gradient boosting (XGBoost), are adopted as the base algorithm. Further, a newly developed Sparrow Search Algorithm (SSA) is hybridized with XGBoost and proposed in the study for the prediction of shrinkage. The study adopts K-fold cross-validation to reduce the risk of overfitting. The results show that the hybridization of SSA-XGBoost outperforms all the algorithms with those without optimization, with the highest performance of R2of 0.91 and RMSE of 79.2 in the testing set. The model is subjected to five-fold cross-validating, ensuring the model is not overfitted. Regarding RMSE, the performance of other models like XGB, LGBM, ETR, and RF is restricted to 102.22,108.38,87.42 and 98.57, respectively. Further, the study incorporated the model explainability behavior and revealed that the curing relative humidity (CRH), steel fiber content (SFS), and sand are the highly influential features for predicting AS. The comprehensive assessment helps understand the parameters influencing AS, making it a helpful tool for researchers to make well-informed decisions.

AbstractLung cancer (LC) is one of the major and risky health defects even the serious cause for death in concurrent era. But no potential drugs even chemotherapeutic agents have been discovered with approval of health safety although some non-toxic biological macromolecules, such as polysaccharides and polysaccharide–protein complexes, have obtained as anti-lung cancer properties. This study conveys the anti-lung cancer properties of 45 polysaccharide derivatives collected from PubChem database. Primarily, the PASS prediction was performed to depict their anti-cancer activity, and 37 compounds showed the desired results. Next, the chemical descriptors, such as HOMO, LUMO, softness, and hardness etc, were calculated through the density functional theory (DFT) for quantum properties. Secondly, the auto molecular docking was executed to delineate the protein-ligand interactions, binding ability and inhibition of active sites of proteins. Additionally, the compounds showed docking score more than −6.40 kcal/mol, and the highest binding affinity was at −10.00 kcal/mol even 15 compounds have higher binding score (-8.6 to −10.0) than approved drugs, Gemcitabine. Succeeding, the most common protein residue, VAL 647, was blocked by ligands for the main protein (1X2J). In addition, five protein’s active sites were determined to make the relative study of protein-ligand interactions. As a result, the target docking against five proteins was performed, and it was found that the targeted docking score as the binding affinity is lower than auto docking. Finally, a comparative study between auto docking and targeted docking was performed for the most common five lung cancer proteins founded in three organisms.Communicated by Ramaswamy H. Sarma

Horizontal directional drilling (HDD) is a widely used trenchless method for underground utility connections. The associated ground settlement triggered by HDD depends on the size, types, and surface texture of pipe, diameter of borehole, and soil conditions. The present study investigates the surface settlement due to the construction of a 1067 mm diameter HDD, which will replace an existing sewer siphon under the SR-60 highway in Chino, California using empirical, and numerical methods. Based on the results obtained from the subsurface investigation, an empirical analysis was conducted first. followed by numerical modeling of the HDD using PLAXIS 2D software. A careful comparison between two different methods indicated closer values of surface settlement between the empirical method (7.3 mm) and the numerical modeling (4.6 mm). In addition, the shape of surface settlement and horizontal settlement curves for the empirical and numerical methods was found to be similar. The minor discrepancy between the two methods resulted as the numerical model can host several soil layers whereas the empirical equation can use only one type of soil. Finally, a parametric study was conducted to evaluate the effect of borehole cover depth, size, and soil parameters on surface settlement. It was observed that soil strength parameters yielded a greater effect on surface movement, whereas modulus of elasticity has a relatively smaller influence with zero contribution from Poisson's ratio.

A study was conducted to identify, discriminate, and quantify the physical characteristics of rat cervix during pregnancy, labor, and both before and after exposure to either prostaglandin (PGE2) or the collagenolytic enzyme matrix metalloprotease-1 (MMP-1). The changes in physical properties of the rat cervix during physiologic ripening was similar to those induced by PGE2 and RU486.

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is [...]

AbstractA metabolic network of energy-sensing molecular pathways drives the biological ageing process. Regulating certain network elements can help decelerate the ageing process and ameliorate ageing associated disorders. Bioactive phytopeptides are a prospective avenue for anti-ageing therapeutics and rejuvenation biotechnology. The present study investigates the potential of therapeutic plant peptides against cellular senescence by targeting three key proteins in the ageing network – target of rapamycin (mTOR), adenosine monophosphate-activated protein kinase (AMPK) and sirtuin 1 (SIRT1). This investigation screened a library of reported bioactive peptides using standard cheminformatic methods including in-silicoADMET, molecular docking, molecular dynamics simulation and molecular mechanics calculation. The retrieved simulation data predict 25 diverse phytopeptides as potential safe and drug-like anti-ageing biologics with half-lives >20 h and bioavailability scores >0.40. The best docked peptide, Cycloleonuripeptide B, exhibited strong binding affinity and stable complex formation with mTOR (-17.5 kCal/mol), SIRT1 (-28.54 kCal/mol) and two active sites in AMPK (-41.8 kCal/mol; −36.0 kCal/mol) during molecular dynamics simulations. The computational study acts as a foundation for future laboratory and clinical research into the potential of repurposing therapeutic phytopeptides against cellular senescence and associated pathophysiology. Communicated by Ramaswamy H. Sarma

AbstractHeritiera fomesis a mangrove plant with a rich history of ethnomedicinal usage against chronic inflammation. Biochemical analyses of H. fomeshave exposed a plethora of bioactive phytochemicals that contribute to this therapeutic effect by perturbing enzymes of a complex inflammatory network mediated by arachidonic acid (AA) metabolism. This study is the first instance of utilizing cheminformatic approaches to elucidate a molecular linkage between these phytochemical interventions and the multi-enzyme AA metabolic network regulation. Analysis of the simulations reflects H. fomesas a functional reservoir of multiple safe and potent natural anti-inflammatory compounds. The investigation suggests two phytocompounds extracted from the plant: a sesquiterpene lactone and a flavone glycoside, as candidate inhibitors of multiple catalytic checkpoints of the inflammatory network. The outcomes of this research act as a primary guideline for future laboratory and clinical testing of anti-inflammatory potentials of H. fomesas an exploitable source of safe and potent drug-like molecules.Communicated by Ramaswamy H. Sarma

Recently, our groups have introduced the notion of optical parametric amplification based on non-Hermitian phase matching wherein the incorporation of loss can lead to gain in this nonlinear optical process. Previous simulation results using second-order nonlinear optical coupled-mode theory have demonstrated the potential of this technique as an alternative to the stringent phase-matching condition, which is often difficult to achieve in semiconductor platforms. Here we fortify this notion for the case of third-order nonlinearity by considering parametric amplification in silicon nanowires and illustrate the feasibility of these devices by employing rigorous finite-difference time-domain analysis using realistic materials and geometric parameters. Particularly, we demonstrate that by systematic control of the optical loss of the idler in a four-wave mixing process, we can achieve efficient unidirectional energy conversion from the pump to the signal component even when the typical phase-matching condition is violated. Importantly, our simulations show that a signal gain of ∼9dB for a waveguide length of a few millimeters is possible over a large bandwidth of several hundreds of nanometers (∼600nm). This bandwidth is nearly 2 orders of magnitude larger than what can be achieved in the conventional silicon-photonics-based four-wave mixing process.

Recycled plastic pin (RPP) has been proved to be an effective and inexpensive solution for shallow slope stabilization. Current practice suggests conducting numerical modeling to find out the desired factor of safety (FS) using RPP in the design of landslide repair. While the slope stability is heavily dependent on soil strength parameters and slope geometry, RPP length and spacing can also play a significant role in reaching the target factor of safety for the highway slope. During this study, a safety prediction model was developed using both statistical and machine learning (ML) approaches to use RPP in slope stabilization. Initially, parametric study was conducted using five different soil strengths, six slope heights, three slope ratios, three RPP lengths, and five RPP spacing configurations. Using the strength reduction techniques of Finite Element Modeling Software PLAXIS 2D, FS was determined for more than 1,000 combinations. Afterwards, a statistical approach was undertaken to determine a safety prediction model containing all possible parameters. Finally, an ML approach was conducted for safety model. The ML approach was found to be more accurate than the classical statistical approach with 85% accuracy of predicting the FS for an RPP reinforced highway slope. The developed model was validated against the values obtained from the numerical modeling, which indicated that the SF obtained from the developed model was in good agreement with those from finite element method (FEM) analysis.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

As with most enterprises, the insurance industry is always on the lookout for the latest technology and ways to apply it to improve business processes. Those of us who've been [...]

Successfully managing litigated claims depends on communication. New technologies that use the Internet can facilitate communication and lead to a more collaborative relationship for all involved. With the ever-increasing pace [...]

Moisture variations significantly influence the strength and stiffness of expansive subgrade soils, shortening the service lives of pavements and increasing the associated maintenance costs. Accurate measurements of soil moisture can be obtained through soil sampling and testing, but the process can be extensive and costly. Empirical models can accurately predict the moisture variations in an expansive subgrade in a shorter period of time, with lower accompanying costs. The objective of the current study was to develop moisture models, using real-time field monitoring data from two hot mix asphalt roads in North Texas. The collected data were analyzed in a statistical environment to solve two first degree Fourier series. The solution produced a moisture variation model that captured variations associated with seasonal effects and temporary variations associated with rainfall. The outputs of this model were within 90% of the values measured on site. Application of the developed models will facilitate noninvasive estimations of the response of soil strength and stiffness properties to variations in moisture.

Calcium alginate (CA) wafer dressings were prepared by lyophilization of hydrogels to deliver ciprofloxacin (CIP) directly to the wound site of infected diabetic foot ulcers (DFUs). The dressings were physically characterized by scanning electron microscopy (SEM), texture analysis (for mechanical and in vitro adhesion properties), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Further, functional properties essential for wound healing, i.e., porosity, in vitro swelling index, water absorption (Aw), equilibrium water content (EWC), water vapor transmission rate (WVTR), evaporative water loss (EWL), moisture content, in vitro drug release and kinetics, antimicrobial activity, and cell viability (MTT assay) were investigated. The wafers were soft, of uniform texture and thickness, and pliable in nature. Wafers showed ideal wound dressing characteristics in terms of fluid handling properties due to high porosity (SEM). XRD confirmed crystalline nature of the dressings and FTIR showed hydrogen bond formation between CA and CIP. The dressings showed initial fast release followed by sustained drug release which can inhibit and prevent re-infection caused by both Gram-positive and Gram-negative bacteria. The dressings also showed biocompatibility (> 85% cell viability over 72 h) with human adult keratinocytes. Therefore, it will be a potential medicated dressing for patients with DFUs infected with drug-resistant bacteria.

We demonstrate a novel differential phase-shift-keying (DPSK) demodulator based on coherent perfect absorption (CPA). Our DPSK demodulator chip device, which incorporates a silicon ring resonator, two bus waveguide inputs, and monolithically integrated detectors, operates passively at a bit rate of 10 Gbps at telecommunication wavelengths, and fits within a mm-scale footprint. Critical coupling is used to achieve efficient CPA by tuning the gap between the ring and bus waveguides. The device has a vertical eye opening of 12.47 mV and a quality factor exceeding 3×10^4. The fundamental principle behind this photonic circuit can be extended to other formats of integrated demodulators.

According to this agreement, citrus food was sent from Pakistan to Russia for the first time in 2021. This first-of-its-kind shipment is a part of the Pakistan-Russia agreement signed in March this year, after the arrival of which the two countries have officially become commercially integrated with each other. After the creation of Central Asian states, Pakistan›s bilateral relations with them have progressed rapidly in other fields, including trade. [Extracted from the article]

Businesses across almost every industry have rapidly adopted Internet-based technology to differentiate themselves from competitors. Internet-based applications for marketing, e-commerce, and customer support have made it easier for companies to [...]

Businesses across almost every industry have rapidly adopted Internet-based technology to differentiate themselves from competitors. Internet-based applications for marketing, e-commerce, and customer support have made it easier for companies to [...]

The ever-increasing complexity of technology decisions presents challenging questions for today's insurance executive. Fortunately, the industry is in the middle of an outsourcing evolution that is redefining operational boundaries, and [...]

The increasing use of GPS as a source of travel survey data has brought with it an increasing need for a reliable source of ground truth. Recently, the main source that has been used for providing ground truth is the prompted recall survey. However, the prompted recall survey is subject to many of the same shortcomings as most self-administered surveys. In this paper, we review some of the common problems encountered in prompted recall surveys. Following this, we describe a recent experiment in using life-logging cameras to record a person's travel, together with GPS, thereby providing a new source of ground truth data.

The recent motion picture 'A.I.' seems to have revived the term 'artificial intelligence' in popular culture. The robots of 'A.I.' explore moviegoers' fascination with the possibility of a future world [...]

Very few studies have been conducted in this part of world to identify relation between maternal serum homocysteine levels and congenital heart disease in their offsprings. With this perspective in mind, this study was carried out.

SUMMARYBacterial pathogens are important targets for detection and identification in medicine, food safety, public health, and security. Bacterial infection is a common cause of morbidity and mortality worldwide. In spite of the availability of antibiotics, these infections are often misdiagnosed or there is an unacceptable delay in diagnosis. Current methods of bacterial detection rely upon laboratory-based techniques such as cell culture, microscopic analysis, and biochemical assays. These procedures are time-consuming and costly and require specialist equipment and trained users. Portable stand-alone biosensors can facilitate rapid detection and diagnosis at the point of care. Biosensors will be particularly useful where a clear diagnosis informs treatment, in critical illness (e.g., meningitis) or to prevent further disease spread (e.g., in case of food-borne pathogens or sexually transmitted diseases). Detection of bacteria is also becoming increasingly important in antibioterrorism measures (e.g., anthrax detection). In this review, we discuss recent progress in the use of biosensors for the detection of whole bacterial cells for sensitive and earlier identification of bacteria without the need for sample processing. There is a particular focus on electrochemical biosensors, especially impedance-based systems, as these present key advantages in terms of ease of miniaturization, lack of reagents, sensitivity, and low cost.

Subsidized energy pricing is a practice that has been followed by almost all the countries, especially developing countries. Despite the requirement of energy subsidy for an economy like one in Bangladesh, it is a parasite that slowly eats up government money and blocks the process of sustainable growth. Bangladesh is not a country which has enough energy resources to sustain for the long run. So, proper consumption and less wastage is a must requirement. Thus, phasing out energy subsidy through a gradual process is the only way to go forward. But, rationalizing energy subsidy has some consequences over the society, economy and environment. So, it is high time to promote rational energy pricing and to prepare for the consequences. The government and policy makers are expected to add up with the steps they are already taking to phase out energy subsidy and thus go for better energy security. In this paper we have discussed the present energy pricing in Bangladesh and the consequence she is going to face if subsidy is phased out. Also we have shown the environmental consequences due to energy subsidy along with a set of proper recommendations for our government to take Bangladesh in the path of sustainable development.

Industrial robot arms are an essential part of automated manufacturing. They automate tasks such as component assembly, welding, light machining and spray painting due to their repeatability, speed and accuracy. The serial industrial robot arm architecture is the most ubiquitous in modern day manufacturing as its technology is mature; the machine architecture provides extreme dexterity and it has a large useful workspace. This architecture however does have some problems; first the machine mass distribution is not efficient as that mass is spread throughout the arm, and the arm itself contains significant inertia, which also leads to some inaccuracy and dynamic vibration problems. The reason for this lies in the location of the motors. This paper focuses on the conceptual design of a novel robotic arm having a hybrid nature, as its architecture departs from both the classic definitions of Serial Kinematics Machines (SKMs) and Parallel Kinematics Machines (PKMs).

The roles of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) during vascular development have been extensively investigated, as has been their role in controlling the responsiveness of the endothelium to exogenous cytokines. However, very little is known about the role of these vascular morphogenic molecules in the pathogenesis of atherosclerosis. Here, we summarize the recent research into angiopoietins in atherosclerosis.

A low cost sensor system is envisaged that has 2 distinct routes for resolution enhancement in position detection. The main area for application of the sensor design would be in the food processing and packaging industry where position resolution of the end effector is in the millimetre to sub-millimetre range. The sensor concept consists of a grid of laser light detectors attached to the stationary base and a grid of lasers attached to the mobile end effector. The photo detectors on the base provide one level for position detection enhancement, that is, the density of the sensor array. The second degree of freedom in improvement comes from the strategic positioning of a set of lasers attached to the end effector.

Heme-oxygenases (HOs) catalyze the conversion of heme into carbon monoxide and biliverdin. HO-1 is induced during hypoxia, ischemia/reperfusion, and inflammation, providing cytoprotection and inhibiting leukocyte migration to inflammatory sites. Although in vitro studies have suggested an additional role for HO-1 in angiogenesis, the relevance of this in vivo remains unknown. We investigated the involvement of HO-1 in angiogenesis in vitro and in vivo. Vascular endothelial growth factor (VEGF) induced prolonged HO-1 expression and activity in human endothelial cells and HO-1 inhibition abrogated VEGF-driven angiogenesis. Two murine models of angiogenesis were used: (1) angiogenesis initiated by addition of VEGF to Matrigel and (2) a lipopolysaccharide (LPS)–induced model of inflammatory angiogenesis in which angiogenesis is secondary to leukocyte invasion. Pharmacologic inhibition of HO-1 induced marked leukocytic infiltration that enhanced VEGF-induced angiogenesis. However, in the presence of an anti-CD18 monoclonal antibody (mAb) to block leukocyte migration, VEGF-induced angiogenesis was significantly inhibited by HO-1 antagonists. Furthermore, in the LPS-induced model of inflammatory angiogenesis, induction of HO-1 with cobalt protoporphyrin significantly inhibited leukocyte invasion into LPS-conditioned Matrigel and thus prevented the subsequent angiogenesis. We therefore propose that during chronic inflammation HO-1 has 2 roles: first, an anti-inflammatory action inhibiting leukocyte infiltration; and second, promotion of VEGF-driven noninflammatory angiogenesis that facilitates tissue repair.

Background: Preeclampsia, a maternal hypertensive disease, is characterized by shallow invasion of the maternal spiral arterioles resulting in hypoxia/reperfusion type insult; however, the molecular mechanism is unknown. The aim of this study was to determine the mechanism of altered oxygen tension or inhibition of phosphatidyl-inositol-3-kinase (PI3K) on trophoblast survival and to investigate the effect of epidermal growth factor (EGF) on maintaining cellular integrity. Materials and Method: We have used flow cytometry, immunoblotting, and fluoroimmunocytochemistry to study apoptosis in a characterized, spontaneously transformed first trimester extravillous-like trophoblast cell line that exhibits many characteristics of in vivo trophoblast. Results: Time-dependent exposure of first trimester extra-villous-like trophoblast to all oxygen tensions tested promoted dissipation of the mitochondrial membrane potential (ψm) and resulted in a significant increase in cell death by 48 hr as determined by dual staining flow cytometry. Western blot analysis revealed expression of cleaved caspase-3 and caspase-9 increased with time with hypoxia and hyperoxia promoting the greatest elevation indicating that longer duration of exposure to a change in oxygen tension causes increased apoptosis via a mitochondrial-mediated pathway. Disruption of the anti-apoptotic PI3K pathway by LY294002 (40 µM), its specific inhibitor, caused further significant dissipation of the ψm(p< 0.01) and cleavage of caspase-3. EGF was able to maintain the ψmand to prevent cleavage of caspase-3 even in the presence of LY294002, indicating that its survival effects were independent of the PI3K pathway. Conclusions: These results suggest that inhibition of the PI3K/Akt pathway can sensitize first-trimester trophoblastlike cells into oxygen-induced cell death and that EGF exerts its anti-apoptotic effect independently of PI3K/Akt.

Angiogenesis is an essential component of endometrial repair and regeneration following menses. Perturbation of this process is associated with menorrhagia, a common gynecological disorder that results in excessive menstrual bleeding. Angiopoietin-1 (Ang-1) promotes vascular maturation via the Tie-2 receptor, while angiopoietin-2 (Ang-2) is its natural antagonist that destabilizes vessels and initiates neovascularization in the presence of vascular endothelial growth factor. To test the hypothesis that menorrhagia arises as a result of poor signal for vascular maturation, we have examined the expression of Ang-1, Ang-2, and Tie-2 in endometrium throughout the menstrual cycle from 30 normal women and 28 patients with menorrhagia. Ribonuclease protection assay and Western blot analysis showed Ang-2 expression was consistently higher than Ang-1 in normal endometrium throughout the cycle. However, with menorrhagia Ang-1 mRNA and protein were not detected or down-regulated, while Ang-2 was observed at similar levels in both normal and menorrhagic endometrium resulting in a greater than a 50% decrease in the ratio of Ang-1 to Ang-2 protein. In situhybridization and immunohistochemical studies supported these findings and revealed cyclical changes in the expression of Ang-1 and Ang-2. These results suggest that the angiopoietin/Tie-2 system promotes vascular remodeling in endometrium and loss of normal Ang-1 expression may contribute to the excessive blood loss observed in menorrhagia.

The known responses of vascular endothelial growth factor (VEGF) are mediated through VEGF receptor-2 (VEGFR-2/KDR) in endothelial cells. However, it is unknown whether VEGFR-1 (Flt-1) is an inert decoy or a signaling receptor for VEGF during physiological or pathological angiogenesis. Here we report that VEGF-stimulated nitric oxide (NO) release is inhibited by blockade of VEGFR-1 and that VEGFR-1 via NO negatively regulates of VEGFR-2-mediated proliferation and promotes formation of capillary networks in human umbilical vein endothelial cells (HUVECs). Inhibition of VEGFR-1 in a murine Matrigel angiogenesis assay induced large aneurysm-like structures. VEGF-induced capillary growth over 14 days was inhibited by anti-VEGFR-2-blocking antibody as determined by reduced tube length between capillary connections (P< 0.0001) in an in vitroangiogenesis assay. In contrast, loss of VEGFR-1 activity with a neutralizing anti-VEGFR-1 antibody resulted in an increase in the accumulation of endothelial cells (P< 0.0001) and a dramatic decrease in the number of capillary connections that were restored by the addition of NO donor. Porcine aortic endothelial (PAE) cells expressing human VEGFR-1 but not VEGFR-2 plated on growth factor-reduced Matrigel rearranged into tube-like structures that were prevented by anti-VEGFR-1 antibody or a cGMP inhibitor. VEGF stimulated NO release from VEGFR-1- but not VEGFR-2-transfected endothelial cells and placenta growth factor-1 stimulated NO release in HUVECs. Blockade of VEGFR-1 increased VEGF-mediated HUVEC proliferation that was inhibited by NO donors, and potentiated by NO synthase inhibitors. These data indicate that VEGFR-1 is a signaling receptor that promotes endothelial cell differentiation into vascular tubes, in part by limiting VEGFR-2-mediated endothelial cell proliferation via NO, which seems to be a molecular switch for endothelial cell differentiation.

Vascular endothelial growth factor (VEGF) receptors are present on nonendothelial cells suggesting that VEGF may mediate nonendothelial effects during organogenesis and tumorigenesis. Here we show that VEGF receptor-1 (VEGFR-1) negatively regulates VEGFR-2-mediated proliferation via nitric oxide (NO) in an epithelial cancer cell line ECV304. Cell proliferation was assessed by [3H]thymidine incorporation, fluorescent-activated cell-sorting analysis, and cell number using a Coulter Counter. Total NO generated by the action of nitric oxide synthase was measured by Seivers NOA 280 Nitric Oxide Chemiluminescence Analyser. VEGF (1 ng/ml) stimulated DNA synthesis and increased ECV304 cell number in a manner that was inhibited by a neutralizing anti-VEGFR-2 antibody. In contrast, VEGF (50 ng/ml) stimulated NO release in a manner that was inhibited by functionally neutralizing anti-VEGFR-1 antibody. Blockage of the VEGFR-1 receptor signal with anti-VEGFR-1 stimulated DNA synthesis and increased cell number. Cell-cycle analysis showed that inhibition of VEGFR-1 increased the transition from G1to S phase whereas inhibition of VEGFR-2 blocked the VEGF-mediated transition from G1to S phase. Finally, the addition of NO donors suppressed both VEGF-mediated proliferation and the increase in growth after blockade of VEGFR-1. Conversely, inhibition of VEGF mediated NO release by nitric oxide synthase inhibitor, l-monomethyl-l-arginine, restored the mitogenic effect of VEGF. These findings identify a dose-dependent reciprocal regulatory mechanism for VEGF via its two receptors. It shows that VEGFR-1 induces cell cytostasis via NO and as such is a suitable target for molecular strategies suppressing tumorigenesis.

Objectives/HypothesisWith some advanced squamous cell carcinomas (SCCs) of the head and neck, chemoradiation therapy may obviate the need for surgical intervention. However, both modalities are known to produce organ to‐icities, and tumor insensitivity remains problematic. Thus there is a clear need for the development of new treatment strategies. Accordingly, preclinical studies to evaluate the use of valrubicin, a contact‐safe, mechanistically novel antitumor agent, combined with low‐dose radiation for the therapy of SCC have been conducted.

Human placental development involves co-ordinated angiogenesis and trophoblast outgrowth that are compromised in intrauterine growth restriction. Adaptive angiogenesis in IUGR placental villi is a result of an imbalance in the orderly progression of the expression profile of vascular endothelial growth factor, placenta growth factor and angiopoietin during placental development. VEGF receptors and the angiopoietin receptor Tie-2 are expressed on trophoblast, and their activation leads to trophoblast proliferation, migration and production of nitric oxide. Thus, these vascular factors act as autocrine regulators of trophoblast behaviour in the development of the utero-/feto-placental circulation, an action independent of their well-established roles in vascular endothelium.

Human placental development involves coordinated angiogenesis and trophoblast outgrowth that are compromised in intrauterine growth restriction (IUGR). As Tie-2(−/−)mice exhibit growth retardation and vascular network malformation, the expression of Tie-2 and its ligands, angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), were investigated in human placenta from normal pregnancies and those complicated by severe IUGR. Ribonucleotide protection assays showed no significant change in the expression of Ang-2 mRNA between gestationally matched normal and IUGR placentas; however, immunoblots revealed that Ang-2 protein was significantly decreased in IUGR, suggesting that this may contribute to the abnormal development of the villous vasculature. In situhybridization studies showed that Ang-1 and Tie-2 were detected in the cyto/syncytiotrophoblast bilayer in first-trimester placenta, whereas Ang-2 mRNA was restricted to the cytotrophoblast, suggesting their role in trophoblast function. At term, Ang-1 mRNA and immunoreactive protein were restricted to the paravascular tissues of the primary stem villi, supporting its role in vessel maturation. In contrast, Ang-2 was expressed throughout the term villous core, perhaps to permit the developing placental vascular network to remain in a state of fluidity. As these studies also revealed that trophoblast, in addition to endothelial cells, expressed Tie-2 receptors, we investigated the potential role of Ang-1/Ang-2 on trophoblast proliferation, migration, and the release of NO. Using spontaneously transformed first-trimester trophoblast cell lines that exhibit cytotrophoblast-like (ED27) and extravillous trophoblast-like (ED77) properties, we show that the addition of Ang-2 (250 ng/ml) stimulated DNA synthesis in ED27trophoblast cells and triggered the release of NO. Ang-1 stimulated trophoblast (ED77) migration in a dose-dependent manner that was inhibited by recombinant Tie-2-FC. These data thus imply, for the first time, a specific role for angiopoietins as regulators of trophoblast behavior in the development of the utero/fetoplacental circulation, an action independent of their well-established roles in vascular endothelium.

Background: Pregnancy is characterized by an inflammatory-like process and this may be exacerbated in preeclampsia. The heme oxygenase (HO) enzymes generate carbon monoxide (CO) that induces blood vessel relaxation and biliverdin that acts as an endogenous antioxidant. Materials and Methods: We examined the expression and localization of HO-1 and HO-2 in normal and preeclamptic placenta using reverse transcription polymerase chain reaction (RT-PCR), RNase protection assay, immunoblotting and immunohistochemistry. In addition, the effect of HO activation on tumor necrosis factor-alpha (TNFα) induced placental damage and on feto-placental circulation was studied. Results: We provide the first evidence for the role of HO as an endogenous placental factor involved with cytoprotection and placental blood vessel relaxation. HO-1 was significantly higher at term, compared with first trimester placentae indicating its role in placental vascular development and regulation. HO-1 predominantly localized in the extravascular connective tissue that forms the perivascular contractile sheath around the developing blood vessels. HO-2 was localized in the capillaries, as well as the villous stroma, with weak staining of trophoblast. Induction of HO-1 caused a significant attenuation of TNFα-mediated cellular damage in placental villous explants, as assessed by lactate dehydrogenase leakage (p&lt; 0.01). HO-1 protein was significantly reduced in placentae from pregnancies complicated with preeclampsia, compared with gestationally matched normal pregnancies. This suggests that the impairment of HO-1 activation may compromise the compensatory mechanism and predispose the placenta to cellular injury and subsequent maternal endothelial cell activation. Isometric contractility studies showed that hemin reduced vascular tension by 61% in U46619-preconstricted placental arteries. Hemininduced vessel relaxation and CO production was inhibited by HO inhibitor, tin protoporphyrin IX. Conclusions: Our findings establish HO-1 as an endogenous system that offers protection against cytotoxic damage in the placenta, identifies the HO-CO pathway to regulate feto-placental circulation and provides a new approach to study the disease of preeclampsia.

Background: Impairment of the fertility in the platelet-activating factor (PAF) receptor transgenic female mice suggests changes in PAF functions can influence uterine receptivity. We hypothesized that vasodilatory actions of PAF in the uterus was exerted by PAF-mediated nitric oxide (NO) release via activation of isoenzyme-specific protein kinase C (PKC). Materials and methods: Inducible and endothelial NOS was shown by Reverse transcription polymerase chain reaction RT-PCR in cDNA synthesized from RNA extract of proliferative and secretory endometrium as well endometrial epithelial cell lines HEC-1B. The effect of WEB2170, NG-monomethyl-l-arginine (l-NMMA) and Ro 31-8220 on PAF mediated NO release by HEC-1B cell was determined. PAF induced translocation of PKCαhr. Before agonist stimulation, in HEC-1B cell and its antagonist effect by Ro 31-8220 was studied by Western immunoblot analysis. PKC isoenzyme regulated by PAF was determined in HEC-1B cell lysate by immunoprecipitation. Results: PAF-evoked a rapid and concentration-dependent biphasic increase in total NO in human HEC-1B endometrial epithelial cell line [as measured by a Sievers NOA 280A NO Chemiluminescent Analyser.] This increase in NO release was attenuated by the PAF receptor antagonist, WEB2170. Inhibition of NO synthesis by NG-monomethyl-l-arginine produced marked dose-dependent attenuation of PAF-mediated NO release, indicating nitric oxide synthase (NOS) activation. PAF-mediated NO release was also inhibited by the PKC inhibitor Ro 31-8220 and by the removal of extracellular calcium, suggesting a dependency on PKC and calcium, respectively. RT-PCR analysis showed expression of inducible NOS and endothelial NOS in human endometrium, myometrium and HEC-1B cells. Western immunoblot analysis showed PKCα, βII and ιwere the principal isozymes present in the HEC-1B cell line and normal endometrium, suggesting that both HEC-1B cells and normal endometrium have similar PKC isozymes. PAF induced the translocation of both PKCαand PKCιwithin the time frame of NO release. The translocation of PKCα, but not PKCι, was susceptible to inhibition by Ro 31-8220 that also inhibited PAF-evoked NO release, suggesting that PKCαis the principal isozyme involved in this process and that eNOS may be a substrate for PKCα. Kinase assays performed using immunoprecipitated PKCαshowed that PAF (1 nM) activated PKCαthat was inhibited by co-incubation with Ro 31-8220 and Ca2+-free medium. Conclusions: This study demonstrates that PAF-stimulated NO release via PKCαin epithelial cells might regulate endometrial functions such as implantation and menstruation.

Vascular endothelial growth factor (VEGF; also known as vascular permeability factor) is a secreted angiogenic growth factor. It is highly specific for endothelial cells, and its receptor, the fms-like tyrosine kinase (flt), has been localized only to endothelial cells in vivo. Here we describe the expression of mRNA encoding flt in human trophoblast as revealed by in situ hybridization. This mRNA is highly expressed in the cytotrophoblast shell and columns and also highly expressed by the extravillous trophoblast (EVT) in the maternal decidua both in the first trimester and at term. The trophoblast-like choriocarcinoma cell line BeWo also expresses this receptor and the related receptor, kinase domain-containing receptor (KDR), which is also a receptor for VEGF. Treatment of the cell line BeWo with VEGF165stimulated 3H-thymidine incorporation and tyrosine phosphorylation of MAP (mitogen-activated protein) kinase in a time- and dose-dependent fashion. This study is the first demonstration of the presence of flt on non-endothelial cells in vivo and suggests a role for VEGF in the growth and differentiation of cytotrophoblast at implantation.

Implantation is characterized by an inflammatory‐like response with expansion of extracellular fluid volume, increased vascular permeability, and vasodilatation. These effects are believed to be mediated at the paracrine level by prostaglandin E2and platelet‐activating factor (PAF), but the cellular mechanism (or mechanisms) remains largely unknown. We demonstrate that PAF receptor (PAF‐R) immunoreactivity and mRNA are detected in proliferative and secretory endometrial glands, however, the responsiveness of endometrium to physiological concentrations of PAF is confined predominantly to the secretory endometrium. Semiquantitative reverse transcription‐polymerase chain reaction revealed that PAF‐R transcript levels were highest in the mid‐late proliferative and late secretory phases of the cycle. Interaction of PAF with its receptor resulted in the rapid release of nitric oxide (NO), increased expression of vascular endothelial growth factor (VEGF), and activation of FAKpp125, a focal adhesion kinase, demonstrating that the PAF‐R is functionally active. Inhibition of NO synthesis by NG‐monomethyl‐L‐arginine produced dose‐dependent attenuation of PAF‐evoked NO release, indicating NOS activation; the dependency of PAF‐evoked NO release on PKC and extracellular Ca2+was confirmed by PKC inhibitor Ro 31–8220 and by the removal of extracellular Ca2+. PAF up‐regulated VEGF gene expression in a concentration‐ and time‐dependent fashion in human endometrial epithelial cell lysates. Transcription of VEGF was rapidly followed by secretion of the protein. These data support our premise that this auto‐coid acts as an angiogenic mediator in the regeneration of the endometrium after menses and as a vasodilator to promote blastocyst attachment during the implantation process.—Ahmed, A., Dearn, S., Shams, M., Li, X. F., Sangha, R. K., Rola‐Pleszczynski Marek, Jiang Jianqiao, Localization, quantification, and activation of plateletactivating factor receptor in human endometrium during the menstrual cycle: PAF stimulates NO, VEGF, and FAKpp125. FASEB J. 12, 831–843 (1998)

Human preimplantation embryos and endometrium secrete platelet-activating factor (PAF). The mechanism of phosphatidylcholine (PC) degradation stimulated by PAF was investigated in endometrial explants prelabeled with [methyl-[3H]choline or preincubated with [3H]butan-1-ol. Analysis of the water-soluble metabolites of PAF-induced PC hydrolysis in secretory endometrium demonstrated that the stimulated generation of [3H]choline (3H]Cho) precedes that of [3H]choline phosphate ([3H]ChoP) and [3H]glycerophosphocholine (3H])GPC). Within 30 sec there was a rapid rise in PAF-induced [3H]Cho generation and by 2 min this had increased to 59.9% ± 10.6% (p< 0.02), with no effect upon [3H]ChoP and [3H]GPC during this period. Both [3H]GPC and [3H]ChoP, however, were increased at a later time point. The slower [3H]ChoP generation may suggest that PCspecific phospholipase C activation as well as delayed [3H]GPC rise may be due to PC-specific phospholipase A2, and lysophospholipase activation. Phospholipase D activity was confirmed by the incorporation of high-specific-activity [3H]butan-1-ol into [3H]phosphatidylbutanol ([3H]PBut). The rapid generation of ([3H]PBut), which paralleled the rise in intracellular [3H]Cho, strongly suggests that PC breakdown is catalyzed by the phospholipase D pathway. it is proposed that PAF induces PC hydrolysis as a consequence of an early phospholipase D-catalyzed breakdown of PC in human secretory endometrium. This may be an alternative source for prostaglandin synthesis and an important pathway essential for long.term activation of local cellular events at the time of implantation.

Placenta growth factor (PlGF) is a growth factor which belongs to the vascular endothelial growth factor (VEGF) family and is known to bind to the fms-like tyrosine kinase receptor (flt-1). Using Western blot analysis a 50 kDa band was identified in placental protein extract which corresponded to PlGF homodimer. Immunoreactive PlGF was localised to the vasculosyncytial membrane and in the media of large blood vessels of the placental villi, while staining within the mesenchyme was weak and diffuse. There was moderate staining for PlGF in discrete cells in the chorion and no staining in the epithelial layer of the amnion. The maternal decidual cells showed strong staining for PlGF immunoreactive protein. PlGF mRNA was predominantly expressed by the vasculosyncytial membrane of villous trophoblast, whilst there was no apparent expression of PlGF mRNA within the villous mesenchyme. These results suggest that PlGF may be an important paracrine factor for vascular endothelial cells in placental angiogenesis and an autocrine mediator of trophoblast function.

Angiogenesis is an essential component of endometrial regeneration after menses in preparation for implantation. Vascular endothelial growth factor (VEGF) is a secreted angiogenic peptide with mitogenic activity specific for endothelial and trophoblast cells. VEGF-immunoreactivity was detected in glandular epithelium throughout the menstrual cycle by immunohistochemistry, but, showed cyclic variation in the stroma and the blood vessels. During the early proliferative phase, strong staining was seen in the glandular epithelial cells while staining in the stroma was confined to a subpopulation of stromal cells and endometrial blood vessels appeared negative. In contrast, very intense staining of the endometrial stromal cells was seen in the mid proliferative endometrium possibly due to increased synthesis of VEGF by oestrogen. In the late proliferative endometrium, staining was seen in the endothelial cells and the perivascular stromal cells around the endometrial blood vessels. The greatest degree of immunostaining of stromal cells was observed in the mid to late proliferative endometrium. Throughout the secretory phase no staining was seen around the endometrial blood vessels and staining of endometrial stromal cells was confined to early secretory endometrium. In the late secretory endometrium only the glands were positive to VEGF antibody. The observed increase in the immunostaining of stroma suggests increased production of VEGF from early to mid and late proliferative endometrium which parallels the increase in the oestradiol levels in the proliferative phase of the menstrual cycle. It is proposed that VEGF may serve as a paracrine mediator of the effects of ovarian steroids on endometrial vascular development.

The placenta undergoes extensive angiogenesis and cellular proliferation to establish adequate blood supply to the fetus. The aim of this study was to compare and contrast the immunolocalization of acidic and basic fibroblast growth factor (FGF) in both first trimester and term placenta and gestational decidua. Human choriocarcinoma cell line JEG-3 were employed as a model of cytotrophoblast and the effect of basic FGF on cell proliferation and phospholipase C and D activation investigated. Basic FGF-immunoreactivity (IR) was detected in or around cytotrophoblast cells and in extravillous trophoblast in first trimester placenta by immunohistochemistry using primary polyclonal rabbit antibodies. Identical staining patterns were produced by acidic FGF antibodies indicating colocalization of acidic FGF and basic FGF. At term, weaker and more diffuse staining was seen in the syncytiotrophoblast surrounding the placenta villi and strong staining was present in the smooth muscle cells of mid and large size placental vessels and in some endothelial cells. Endothelial cells and extravillous trophoblast stained strongly within the decidua at first trimester, whereas the glandular epithelium was weakly stained. Basic FGF induced [3H]thymidine incorporation in JEG-3 cells in a dose dependent manner and caused an increase in inosital phosphate accumulation in cells pre-labelled with myo-[3H]inosital at similar concentrations, suggesting a role of phospholipase C in JEG-3 cell proliferation. However, basic FGF failed to stimulate phospholipase D activity in cells pre-labelled with [3H]myristic acid. The detection of acid FGF and basic FGF on both maternal and fetal side of the placenta during early pregnancy suggests a role for FGF in angiogenesis, whereas localisation of the growth factor at term, when extensive angiogenesis has diminished, would indicate that FGF may be associated with more differentiated functions of the trophoblast. The nuclear localization of basic FGF in dividing but not non-dividing placental cells together with the effect of basic FGF on JEF-3 cells, strongly supports a role for basic FGF in cytotrophoblast proliferation in vivo.