Publications by authors named "Amjad Farooq"

93 Publications

Health risks of arsenic buildup in soil and food crops after wastewater irrigation.

Sci Total Environ 2021 Jun 21;772:145266. Epub 2021 Jan 21.

Department of Environmental Sciences, COMSATS University Islamabad, Vehari Campus, 61100, Pakistan.

Despite considerable research of arsenic (As) level in ground/drinking water of Pakistan, scarce data is available regarding irrigation water contamination by As and associated health risks. The municipal wastewater is routinely applied for soil irrigation in peri-urban agriculture of the country. Since the wastewater composition/contamination and its allied consequences greatly vary in different areas, therefore, it is imperative to check the possible health risks in areas where untreated wastewater is being applied for food crop production. This study analyzed potential health hazards of As-buildup in soil and food plants irrigated with municipal wastewater growing under natural conditions. Sixteen wastewater irrigation locations were selected in District Vehari. From these sites, a total of 16 wastewater samples, 108 soil samples and 65 plant samples were collected for As analysis. Total As contents in wastewater (5.3-63.6 μg/L), soil (1.4-19.6 mg/kg) and plants (0-6.5 mg/kg) greatly varied with sampling location, soil depths and plant type. Based on total As contents in edible tissues, risk assessment parameters, especially cancer risk factor, showed possible health risks (> 0.0001) for wheat crops for children while no risks for other food crops. The use of multiple and diversified food crops is recommended in the study area to minimize the possible risk of As exposure and poisoning. The study also anticipates some future viewpoints considering the on-ground situation of wastewater use, possible exposure of metal(loid)s to human and associated health concerns at local and global scale.
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http://dx.doi.org/10.1016/j.scitotenv.2021.145266DOI Listing
June 2021

Deep Ensemble Model for Classification of Novel Coronavirus in Chest X-Ray Images.

Comput Intell Neurosci 2021 5;2021:8890226. Epub 2021 Jan 5.

Department of Computer Science, University of Engineering and Technology, Lahore 54890, Pakistan.

The novel coronavirus, SARS-CoV-2, can be deadly to people, causing COVID-19. The ease of its propagation, coupled with its high capacity for illness and death in infected individuals, makes it a hazard to the community. Chest X-rays are one of the most common but most difficult to interpret radiographic examination for early diagnosis of coronavirus-related infections. They carry a considerable amount of anatomical and physiological information, but it is sometimes difficult even for the expert radiologist to derive the related information they contain. Automatic classification using deep learning models can help in better assessing these infections swiftly. Deep CNN models, namely, MobileNet, ResNet50, and InceptionV3, were applied with different variations, including training the model from the start, fine-tuning along with adjusting learned weights of all layers, and fine-tuning with learned weights along with augmentation. Fine-tuning with augmentation produced the best results in pretrained models. Out of these, two best-performing models (MobileNet and InceptionV3) selected for ensemble learning produced accuracy and FScore of 95.18% and 90.34%, and 95.75% and 91.47%, respectively. The proposed hybrid ensemble model generated with the merger of these deep models produced a classification accuracy and FScore of 96.49% and 92.97%. For test dataset, which was separately kept, the model generated accuracy and FScore of 94.19% and 88.64%. Automatic classification using deep ensemble learning can help radiologists in the correct identification of coronavirus-related infections in chest X-rays. Consequently, this swift and computer-aided diagnosis can help in saving precious human lives and minimizing the social and economic impact on society.
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http://dx.doi.org/10.1155/2021/8890226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805527PMC
January 2021

Cadmium Partitioning, Physiological and Oxidative Stress Responses in Marigold (Calendula calypso) Grown on Contaminated Soil: Implications for Phytoremediation.

Bull Environ Contam Toxicol 2020 Aug 13;105(2):270-276. Epub 2020 Jul 13.

Department of Horticulture, Muhammad Nawaz Shareef University of Agriculture, Multan, Pakistan.

Marigold (Calendula calypso) is a multipurpose ornamental plant, but its cadmium (Cd) tolerance and phytoremediation potential is unknown. The proposed study was carried out to unravel Cd partitioning, physiological and oxidative stress responses of C. calypso grown under Cd stress. Plants were grown for four months in pots having different soil Cd levels: 0, 25, 50, 75, and 100 mg kg soil. Plant growth, biomass, photosynthetic pigments, leaf water contents, stomatal conductance, and membrane stability index were not decreased at 25 mg kg Cd. At higher levels of Cd stress, activities of antioxidant enzymes (SOD, APX, CAT, POD) increased to mitigate HO and lipid peroxidation. Cadmium uptake in plants increased with increasing soil Cd levels, and roots accumulated a greater portion of Cd, followed by shoots and flowers, respectively. On the basis of Cd accumulation and its tolerance, it was determined that C. calypso can be successfully grown for phytostabilization of Cd contaminated soils.
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http://dx.doi.org/10.1007/s00128-020-02934-6DOI Listing
August 2020

Cellulose from sources to nanocellulose and an overview of synthesis and properties of nanocellulose/zinc oxide nanocomposite materials.

Int J Biol Macromol 2020 Jul 19;154:1050-1073. Epub 2020 Mar 19.

College of Textiles, Donghua University, Shanghai 201620, China; Innovation Center for Textile Science and Technology, Donghua University, Shanghai 201620, China. Electronic address:

Recently, environmental and ecological concerns are increasing due to the usage of petroleum-based products so the synthesis of ultra-fine chemicals and functional materials from natural resources is drawing a tremendous level of attention. Nanocellulose, a unique and promising natural material extracted from native cellulose, may prove to be most ecofriendly materials that are technically and economically feasible in modern times, minimizing the pollution generation. Nanocellulose has gained tremendous attention for its use in various applications, due to its excellent special surface chemistry, physical properties, and remarkable biological properties (biodegradability, biocompatibility, and non-toxicity). Various types of nanocellulose, viz. cellulose nanofibrils (CNFs), cellulose nanocrystals (CNCs), and bacterial nanocellulose (BNC), are deeply introduced and compared in this work in terms of sources, production, structures and properties. The metal and metal oxides especially zinc oxide nanoparticles (ZnO-NPs) are broadly used in various fields due to the diversity of functional properties such as antimicrobial and ultraviolet (UV) properties. Thus, the advancement of nanocellulose and zinc oxide nanoparticles (ZnO-NPs)-based composites materials are summarized in this article in terms of the preparation methods and remarkable properties with the help of recent knowledge and significant findings (especially from the past six years reports). The nanocellulose materials complement zinc oxide nanoparticles, where they impart their functional properties to the nanoparticle composites. As a result hybrid nanocomposite containing nanocellulose/zinc oxide composite has shown excellent mechanical, UV barrier, and antibacterial properties. The nanocellulose based hybrid nanomaterials have huge potential applications in the area of food packaging, biopharmaceuticals, biomedical, and cosmetics. Thus the functional composite materials containing nanocellulose and zinc oxide will determine the potential biomedical application for nanocellulose.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.03.163DOI Listing
July 2020

Study of land cover/land use changes using RS and GIS: a case study of Multan district, Pakistan.

Environ Monit Assess 2019 Dec 2;192(1). Epub 2019 Dec 2.

Department of Environmental Sciences, COMSATS University Islamabad, Vehari Campus, Vehari, Punjab, 61100, Pakistan.

Water and land both are limited resources. Current management strategies are facing multiple challenges to meet food security of an increasing population in numerous South Asian countries, including Pakistan. The study of land cover/land use changes (LCLUC) and land surface temperature (LST) is important as both provide critical information for policymaking of natural resources. We spatially examined LCLU and LST changes in district Multan, Pakistan, and its impacts on vegetation cover and water during 1988 to 2017. The LCLUC indicate that rice and sugarcane had less volatility of change in comparison with both cotton and wheat. Producer's accuracy (PA) is the map accuracy (the producer of map), but user's accuracy (UA) is the accuracy from the point of view of a map user, not the map maker. Average overall producer's and user's accuracy for the region was 85.7% and 87.7% for Rabi (winter) and Kharif (summer) seasons, respectively. The results of this study showed that 'built-up area' increased with 7.2% of all the classes during 1988 to 2017 in the Multan district. Anthropogenic activities decreased the vegetation, leading to an increase in LST in study area. Changes on LCLU and LST during the last 30 years have shown that vegetation pattern has changed and temperature has increased in the Multan district.
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http://dx.doi.org/10.1007/s10661-019-7959-1DOI Listing
December 2019

Novel variant p.E269K confirms causative role of PLS1 mutations in autosomal dominant hearing loss.

Clin Genet 2019 12 27;96(6):575-578. Epub 2019 Aug 27.

John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, Florida.

Auditory reception relies on the perception of mechanical stimuli by stereocilia and its conversion to electrochemical signal. Mechanosensory stereocilia are abundant in actin, which provides them with structural conformity necessary for perception of auditory stimuli. Out of three major classes of actin-bundling proteins, plastin 1 encoded by PLS1, is highly expressed in stereocilia and is necessary for their regular maintenance. A missense PLS1 variant associated with autosomal dominant hearing loss (HL) in a small family has recently been reported. Here, we present another PLS1 missense variant, c.805G > A (p.E269K), in a Turkish family with autosomal dominant non-syndromic HL confirming the causative role of PLS1 mutations in HL. We propose that HL due to the p.E269K variant is from the loss of a stable PLS1-ACTB interaction.
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http://dx.doi.org/10.1111/cge.13626DOI Listing
December 2019

Interplay between HGAL and Grb2 proteins regulates B-cell receptor signaling.

Blood Adv 2019 08;3(15):2286-2297

Division of Oncology-Hematology, Department of Medicine, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami, FL.

Human germinal center (GC)-associated lymphoma (HGAL) is an adaptor protein expressed in GC B cells. HGAL regulates cell motility and B-cell receptor (BCR) signaling, processes that are central for the successful completion of the GC reaction. Herein, we demonstrate phosphorylation of HGAL by Syk and Lyn kinases at tyrosines Y80, Y86, Y106Y107, Y128, and Y148. The HGAL YEN motif (amino acids 107-109) is similar to the phosphopeptide motif pYXN used as a binding site to the growth factor receptor-bound protein 2 (Grb2). We demonstrate by biochemical and molecular methodologies that HGAL directly interacts with Grb2. Concordantly, microscopy studies demonstrate HGAL-Grb2 colocalization in the membrane central supramolecular activation clusters (cSMAC) following BCR activation. Mutation of the HGAL putative binding site to Grb2 abrogates the interaction between these proteins. Further, this HGAL mutant localizes exclusively in the peripheral SMAC and decreases the rate and intensity of BCR accumulation in the cSMAC. Furthermore, we demonstrate that Grb2, HGAL, and Syk interact in the same complex, but Grb2 does not modulate the effects of HGAL on Syk kinase activity. Overall, the interplay between the HGAL and Grb2 regulates the magnitude of BCR signaling and synapse formation.
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http://dx.doi.org/10.1182/bloodadvances.2018016162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693015PMC
August 2019

Assessment of Lagrangian particle dispersion model "LAPMOD" through short range field tracer test in complex terrain.

J Environ Radioact 2019 Sep 13;205-206:34-41. Epub 2019 May 13.

Pakistan Institute of Engineering and Applied Sciences (PIEAS), Nilore, Islamabad, Pakistan.

In this paper the mesoscale application of the 3D Lagrangian particle dispersion model LAPMOD has been assessed for a field tracer test performed in a short-range complex terrain. The meteorological input was provided through the diagnostic model CALMET, the meteorological pre-processor of the CALPUFF model. The CALMET/LAPMOD coupled system was used to simulate the hourly averaged ground level concentration at 47 discrete receptors. The LAPMOD model has a general tendency to slightly underestimate the hourly averaged ground-level concentrations. A Q-Q plot shows that the predicted concentration distribution has a good comparison with observed one. The Robust Highest Concentration (RHC) indicates that the LAPMOD model slightly underestimates the simulated peak concentration in short-term release conditions. The Fractional Bias (FB), Normalized Mean Squared Error (NMSE), Factor of Two (FAC2), Factor of Four (FAC4) and Factor of Exceedance (FOEX) statistical indices were calculated. The predicted results by LAPMOD are generally in good agreement with observed ones and the model is justified for the use in complex terrain for short-term near-field applications.
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http://dx.doi.org/10.1016/j.jenvrad.2019.04.015DOI Listing
September 2019

Assessment of arsenic exposure by drinking well water and associated carcinogenic risk in peri-urban areas of Vehari, Pakistan.

Environ Geochem Health 2020 Jan 3;42(1):121-133. Epub 2019 May 3.

Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, Faisalabad, 38040, Pakistan.

Arsenic (As) is a highly toxic and carcinogenic element. It has received considerable consideration worldwide in recent years due to its highest toxicity to human, and currently, high concentrations observed in the groundwater. Some recent media and research reports also highlighted possible As contamination of groundwater systems in Pakistan. However, there is a scarcity of data about As contents in groundwater in different areas/regions of the country. Consequently, the current study estimated the As concentration in the groundwater used for drinking purpose in 15 peri-urban sites of district Vehari, Pakistan. In total, 127 groundwater samples were collected and examined for As contents in addition to physicochemical characteristics such as temperature, electrical conductivity, pH, total soluble salts, chloride, carbonates, bicarbonates, sodium, potassium, lithium, calcium and barium. Results indicated that the groundwater samples were not fully fit for drinking purposes with several parameters, especially the alarming levels of As (mean As: 46.9 µg/L). It was found that 83% groundwater samples of peri-urban sites in district Vehari have As concentration greater than WHO lower permissible limit (10 µg/L). The risk assessment parameters (mean hazard quotient: 3.9 and mean cancer risk: 0.0018) also showed possible carcinogenic and non-carcinogenic risks associated with ingestion of As-contaminated groundwater at peri-urban sites. Based on the findings, it is anticipated that special monitoring and management of groundwater is necessary in the studied area in order to curtail the health risks associated with the use of As-contaminated drinking water. Moreover, appropriate remediation and removal of As from groundwater is also imperative for the study area before being used for drinking purpose to avoid As exposure and related risks to the local community.
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http://dx.doi.org/10.1007/s10653-019-00306-6DOI Listing
January 2020

Association Of Non-Alcoholic Fatty Liver Disease With Serum Uric Acid.

J Ayub Med Coll Abbottabad 2019 Jan-Mar;31(1):64-66

Department of Surgery, Ayub Medical College Abbottabad, Pakistan.

Background: Non-alcoholic fatty liver disorder is a clinicopathological condition, characterized by macro vesicular steatosis in hepatic cells and metabolic stress related disorders without other causes of chronic hepatic disease. Uric acid is basically a heterocyclic compound of hydrogen, carbon, oxygen and nitrogen. Uric acid is the metabolic end product of purine metabolism. Hyperuricemia is considered to be related with the causes responsible for the production of metabolic syndrome. It may cause gout, impaired renal function, hypertension, hypertriglyceridemia, obesity and diabetes mellitus. The objective of study is to investigate the association of non-alcoholic fatty liver disease (NAFLD) and serum uric acid level.

Methods: This cross-sectional study was conducted at Ayub Teaching institute Abbottabad. According to predesigned questionnaire and informed consent, 100 subjects between ages 40-50 years were selected for the study. Data collected and analysis done by SPSS version 20.

Results: It was observed that 20 (40%) subjects developed NAFLD as compared to 30(60%) of the subjects with normal serum uric acid level. Chi-square test was applied and values found to be significant (p=0.013).

Conclusions: Our study shows association of NAFLD with serum uric acid level.
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May 2019

Comparison of the effects of gamma or sonochemical irradiation of carbon nanotubes and the influence on the mechanical and dielectric properties of chitosan nanocomposites.

Ultrason Sonochem 2019 Jun 28;54:241-249. Epub 2019 Jan 28.

Pakistan Institute of Engineering and Applied Sciences, Islamabad, Pakistan.

Chitosan-carbon nanotube (Chi-CNT) composite materials have been prepared with CNTs that were surface treated using either dilute acid combined with 20 kHz ultrasound or gamma-irradiation in air. The mechanical and dielectric properties have been measured and compared. Both modification methods gave nanocomposites with much improved tensile properties over native chitosan. The sonochemically treated samples were stronger with higher tensile strength but at the expense of lower elasticity and extensibility than found when γ-irradiation was used. Impedance spectra showed differences in the polymer chain transitions and in the conduction mechanisms within the nanocomposites. The results correlated well with previous work suggesting that the two modification techniques result in CNT surfaces with higher polarity. This enhances interfacial interactions with the chitosan matrix although the extent of functionalisation was greater in the sonochemical case. This work demonstrates that sonochemical modification under mild conditions is a useful method for modifying CNTs for inclusion in nanocomposite materials. However, the resulting material properties depend on the level of treatment so that the sonochemical conditions need to be carefully evaluated and controlled if the effects are to be optimised.
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http://dx.doi.org/10.1016/j.ultsonch.2019.01.033DOI Listing
June 2019

Dysfunction of , encoding the GRB2-related adaptor protein, is linked to sensorineural hearing loss.

Proc Natl Acad Sci U S A 2019 01 4;116(4):1347-1352. Epub 2019 Jan 4.

John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136;

We have identified a variant (c.311A>T; p.Gln104Leu) cosegregating with autosomal recessive nonsyndromic deafness in two unrelated families. encodes a member of the highly conserved growth factor receptor-bound protein 2 (GRB2)/Sem-5/drk family of proteins, which are involved in Ras signaling; however, the function of the growth factor receptor-bound protein 2 (GRB2)-related adaptor protein (GRAP) in the auditory system is not known. Here, we show that, in mouse, is expressed in the inner ear and the protein localizes to the neuronal fibers innervating cochlear and utricular auditory hair cells. Downstream of receptor kinase (), the homolog of human , is expressed in Johnston's organ (JO), the fly hearing organ, and the loss of in JO causes scolopidium abnormalities. mutant flies present deficits in negative geotaxis behavior, which can be suppressed by human wild-type but not mutant GRAP. Furthermore, drk specifically colocalizes with synapsin at synapses, suggesting a potential role of such adaptor proteins in regulating actin cytoskeleton dynamics in the nervous system. Our findings establish a causative link between mutation and nonsyndromic deafness and suggest a function of GRAP/drk in hearing.
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http://dx.doi.org/10.1073/pnas.1810951116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347722PMC
January 2019

Effect of salinity on cadmium tolerance, ionic homeostasis and oxidative stress responses in conocarpus exposed to cadmium stress: Implications for phytoremediation.

Ecotoxicol Environ Saf 2019 Apr 29;171:146-153. Epub 2018 Dec 29.

Department of Environmental Sciences, COMSATS University Islamabad, Vehari Campus, Pakistan.

Contamination of soil with salinity and Cd negatively affects growth and productivity of plants. The proposed study has been planned to explore the effects of salinity on Cd uptake, tolerance and phytoremediation potential of conocarpus (Conocarpus erectus L.). One-month-old uniform plants of conocarpus were exposed to 0, 8.9, 44.5, 89 and 178 µM Cd alone or in combination with 0, 100 and 200 mM NaCl in Hoagland's nutrient solution. Results revealed that shoot and root biomasses, leaf water content and pigment content decreased more in response to combination of Cd and salinity compared to Cd alone. The Na and Cl concentrations in shoot and root were not affected by Cd alone, but increased in Cd + salinity treatments. The K concentration decreased by Cd alone as well as Cd combination with salinity. Plant Cd uptake increased in the presence of salinity but its translocation from root to shoot remained unaffected. Exposure of plants to Cd alone and Cd + salinity caused oxidative stress via overproduction of HO and inducing lipid peroxidation. The activities of antioxidant enzymes such as SOD, CAT, POD and APX increased to mitigate this oxidative stress. It is concluded that the tolerance of conocarpus against Cd stress is decreased in the presence of salinity due to increased uptake of toxic ions and intensification of oxidative stress. Moreover, the Cd uptake behavior of this tree indicates its suitability for phytostabilization of Cd contaminated saline and non-saline soils.
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http://dx.doi.org/10.1016/j.ecoenv.2018.12.077DOI Listing
April 2019

Spectral Dependence of Degradation under Ultraviolet Light in Perovskite Solar Cells.

ACS Appl Mater Interfaces 2018 Jul 25;10(26):21985-21990. Epub 2018 Jun 25.

Institute of Microstructure Technology , Karlsruhe Institute of Technology , Hermann-von-Helmholtz-Platz 1 , 76344 Eggenstein-Leopoldshafen , Germany.

Perovskite solar cells (PSCs) demonstrate excellent power conversion efficiencies (PCEs) but face severe stability challenges. One key degradation mechanism is exposure to ultraviolet (UV) light. However, the impact of different UV bands is not yet well established. Here, we systematically study the stability of PSCs on the basis of a methylammonium lead iodide (CHNHPbI) absorber exposed to (i) 310-317 (UV-B range) and (ii) 360-380 nm (UV-A range), under accelerated conditions. We demonstrate that the investigated UV-B band is detrimental to the stability of PSCs, resulting in PCE degradation by more than 50% after an exposure period >1700 sun-hours. This finding is valid for architectures with a range of electron transport layers, including SnO, compact-TiO, electron-beam TiO, and nanoparticle-TiO. We also show that photodegradation is apparent for high, as well as for low illumination intensities of UV-B light, but not for illumination with UV-A wavelengths. Finally, we show that degradation of PSCs is preventable at the cost of a small fraction of photocurrent by using UV-filtering or luminescent downshifting layers.
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http://dx.doi.org/10.1021/acsami.8b03024DOI Listing
July 2018

SCO2 mutations cause early-onset axonal Charcot-Marie-Tooth disease associated with cellular copper deficiency.

Brain 2018 03;141(3):662-672

Department of Neurology, Carver College of Medicine, University of Iowa, Iowa City, USA.

Recessive mutations in the mitochondrial copper-binding protein SCO2, cytochrome c oxidase (COX) assembly protein, have been reported in several cases with fatal infantile cardioencephalomyopathy with COX deficiency. Significantly expanding the known phenotypic spectrum, we identified compound heterozygous variants in SCO2 in two unrelated patients with axonal polyneuropathy, also known as Charcot-Marie-Tooth disease type 4. Different from previously described cases, our patients developed predominantly motor neuropathy, they survived infancy, and they have not yet developed the cardiomyopathy that causes death in early infancy in reported patients. Both of our patients harbour missense mutations near the conserved copper-binding motif (CXXXC), including the common pathogenic variant E140K and a novel change D135G. In addition, each patient carries a second mutation located at the same loop region, resulting in compound heterozygote changes E140K/P169T and D135G/R171Q. Patient fibroblasts showed reduced levels of SCO2, decreased copper levels and COX deficiency. Given that another Charcot-Marie-Tooth disease gene, ATP7A, is a known copper transporter, our findings further underline the relevance of copper metabolism in Charcot-Marie-Tooth disease.
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http://dx.doi.org/10.1093/brain/awx369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837310PMC
March 2018

Phosphorylation of Tyr188 in the WW domain of YAP1 plays an essential role in YAP1-induced cellular transformation.

Cell Cycle 2016 Sep 18;15(18):2497-505. Epub 2016 Jul 18.

a Department of Cancer Genetics , Roswell Park Cancer Institute , Buffalo , NY , USA.

The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP1, a transcriptional co-activator amplified in mouse and human cancers where it promotes epithelial-to-mesenchymal transition (EMT) and malignant transformation. The Hippo tumor suppressor pathway has been suggested to inhibit the YAP1 function through serine phosphorylation-induced cytoplasmic retention and degradation. Here we report that the tyrosine188 (Y188) site of YAP1 isoform with 2 WW domains (known as YAP1-2) plays an important role in YAP1-induced cellular transformation. IP-Mass Spectrometry analysis of YAP1 identified the phosphorylation of Y188 but not other tyrosine residues. In contrast to the aberrant 3D acinus formation observed in YAP1-WT transduced cells, overexpression of YAP1-Y188F (non-phosphorylated mimic) displayed normal 3D structures. In addition, knockdown of the endogenous YAP1 in MDA-MB231 breast cancer cells inhibited cell proliferation and migration, which were then successfully rescued by the exogenous YAP1-WT and YAP1-Y188E but not Y188F. Mechanistically, we also demonstrated that YAP1-Y188F had a higher affinity to the upstream negative regulator PTPN14 and was extensively localized in the cytoplasm. Since the Y188 is located in the conserved aromatic core of the WW domain of YAP1, our finding has a wide implication for WW domain signaling in general, where Y phosphorylation may act as a common positive regulator of the complex formation via WW domains. In summary, our results indicate that tyrosine 188 plays an important role in the YAP1-induced cellular transformation and its phosphorylation may intriguingly serve as a positive indicator of YAP1 activation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026814PMC
http://dx.doi.org/10.1080/15384101.2016.1207836DOI Listing
September 2016

Characterization of ATPase Activity of P2RX2 Cation Channel.

Front Physiol 2016 24;7:186. Epub 2016 May 24.

Department of Otolaryngology, University of Miami Miller School of MedicineMiami, FL, USA; Department of Biochemistry, University of Miami Leonard M. Miller School of MedicineMiami, FL, USA; Department of Otolaryngology, Central South University, Xiangya HospitalChangsha, China.

P2X purinergic receptors are plasma membrane ATP-dependent cation channels that are broadly distributed in the mammalian tissues. P2RX2 is a modulator of auditory sensory hair cell mechanotransduction and plays an important role in hair cell tolerance to noise. In this study, we demonstrate for the first time in vitro and in cochlear neuroepithelium, that P2RX2 possesses the ATPase activity. We observed that the P2RX2 V60L human deafness mutation alters its ability to bind ATP, while the G353R has no effect on ATP binding or hydrolysis. A non-hydrolysable ATP assay using HEK293 cells suggests that ATP hydrolysis plays a significant role in the opening and gating of the P2RX2 ion channel. Moreover, the results of structural modeling of the molecule was in agreement with our experimental observations. These novel findings suggest the intrinsic ATPase activity of P2RX2 and provide molecular insights into the channel opening.
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http://dx.doi.org/10.3389/fphys.2016.00186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878533PMC
June 2016

ROR1 is essential for proper innervation of auditory hair cells and hearing in humans and mice.

Proc Natl Acad Sci U S A 2016 May 9;113(21):5993-8. Epub 2016 May 9.

John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136; Department of Otolaryngology, Miller School of Medicine, University of Miami, Miami, FL 33136; Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136

Hair cells of the inner ear, the mechanosensory receptors, convert sound waves into neural signals that are passed to the brain via the auditory nerve. Little is known about the molecular mechanisms that govern the development of hair cell-neuronal connections. We ascertained a family with autosomal recessive deafness associated with a common cavity inner ear malformation and auditory neuropathy. Via whole-exome sequencing, we identified a variant (c.2207G>C, p.R736T) in ROR1 (receptor tyrosine kinase-like orphan receptor 1), cosegregating with deafness in the family and absent in ethnicity-matched controls. ROR1 is a tyrosine kinase-like receptor localized at the plasma membrane. At the cellular level, the mutation prevents the protein from reaching the cellular membrane. In the presence of WNT5A, a known ROR1 ligand, the mutated ROR1 fails to activate NF-κB. Ror1 is expressed in the inner ear during development at embryonic and postnatal stages. We demonstrate that Ror1 mutant mice are severely deaf, with preserved otoacoustic emissions. Anatomically, mutant mice display malformed cochleae. Axons of spiral ganglion neurons show fasciculation defects. Type I neurons show impaired synapses with inner hair cells, and type II neurons display aberrant projections through the cochlear sensory epithelium. We conclude that Ror1 is crucial for spiral ganglion neurons to innervate auditory hair cells. Impairment of ROR1 function largely affects development of the inner ear and hearing in humans and mice.
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http://dx.doi.org/10.1073/pnas.1522512113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889368PMC
May 2016

A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model.

Vaccine 2015 Sep 1;33(38):4798-806. Epub 2015 Aug 1.

Department of Microbiology and Immunology, Miami Center for AIDS Research, and the Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, USA. Electronic address:

Vaccination with tumor-associated antigens can induce cancer-specific CD8+ T cells. A recent improvement has been the targeting of antigen to dendritic cells (DC) using antibodies that bind DC surface molecules. This study explored the use of multi-trimers of CD40L to target the gp100 melanoma tumor antigen to DC. The spontaneously-multimerizing gene Surfactant Protein D (SPD) was used to fuse gp100 tumor antigen and CD40L, creating the recombinant protein SPD-gp100-CD40L. This "third generation" DC-targeting vaccine was designed to both target antigen to DC and optimally activate dendritic cells by aggregating CD40 trimers on the DC membrane surface. SPD-gp100-CD40L expressed as a 110kDa protein. Analytical light scattering analysis gave elution data corresponding to 4-trimer and multi-trimer SPD-gp100-CD40L oligomers. The protein was biologically active on dendritic cells and induced CD40-mediated NF-κB signaling. DNA vaccination with SPD-gp100-CD40L plasmid, together with plasmids encoding IL-12p70 and GM-CSF, significantly enhanced survival and inhibited tumor growth in a B16-F10 melanoma model. Expression of gp100 and SPD-CD40L as separate molecules did not enhance survival, highlighting the requirement to encode gp100 within SPD-CD40L for optimal vaccine activity. These data support a model where DNA vaccination with SPD-gp100-CD40L targets gp100 to DC in situ, induces activation of these DC, and generates a protective anti-tumor response when given in combination with IL-12p70 and GM-CSF plasmids.
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http://dx.doi.org/10.1016/j.vaccine.2015.07.081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562804PMC
September 2015

Structural and Functional Diversity of Estrogen Receptor Ligands.

Authors:
Amjad Farooq

Curr Top Med Chem 2015 ;15(14):1372-84

Department of Biochemistry & Molecular Biology, Leonard Miller School of Medicine, University of Miami, Miami, FL 33136, USA.

Estrogen receptors, comprised of ERα and ERβ isoforms in mammals, act as ligandmodulated transcription factors and orchestrate a plethora of cellular functions from sexual development and reproduction to metabolic homeostasis. Herein, I revisit the structural basis of the binding of ERα to DNA and estradiol in light of the recent discoveries and emerging trends in the field of nuclear receptors. A particular emphasis of this review is on the chemical and structural diversity of an everincreasing repertoire of physiological, environmental and synthetic ligands of estrogen receptors that ultimately modulate their interactions with cognate DNA located within the promoters of estrogenresponsive genes. In particular, modulation of estrogen receptors by small molecule ligands represents an important therapeutic goal toward the treatment of a wide variety of human pathologies including breast cancer, cardiovascular disease, osteoporosis and obesity. Collectively, this article provides an overview of a wide array of small organic and inorganic molecules that can fine-tune the physiological function of estrogen receptors, thereby bearing a direct impact on human health and disease.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604006PMC
http://dx.doi.org/10.2174/1568026615666150413154841DOI Listing
August 2016

Allostery mediates ligand binding to WWOX tumor suppressor via a conformational switch.

J Mol Recognit 2015 Apr 19;28(4):220-31. Epub 2015 Feb 19.

Department of Biochemistry and Molecular Biology, Leonard Miller School of Medicine University of Miami, Miami, FL, 33136, USA.

While being devoid of the ability to recognize ligands itself, the WW2 domain is believed to aid ligand binding to the WW1 domain in the context of a WW1-WW2 tandem module of WW domain-containing oxidoreductase (WWOX) tumor suppressor. In an effort to test the generality of this hypothesis, we have undertaken here a detailed biophysical analysis of the binding of WW domains of WWOX alone and in the context of the WW1-WW2 tandem module to an array of putative proline-proline-x-tyrosine (PPXY) ligands. Our data show that while the WW1 domain of WWOX binds to all ligands in a physiologically relevant manner, the WW2 domain does not. Moreover, ligand binding to the WW1 domain in the context of the WW1-WW2 tandem module is two-to-three-fold stronger than when treated alone. We also provide evidence that the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. Of particular note is the observation that the physical association of the WW2 domain with WW1 blocks access to ligands. Consequently, ligand binding to the WW1 domain not only results in the displacement of the WW2 lid but also disrupts the physical association of WW domains in the liganded conformation. Taken together, our study underscores a key role of allosteric communication in the ability of the WW2 orphan domain to chaperone physiological action of the WW1 domain within the context of the WW1-WW2 tandem module of WWOX.
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http://dx.doi.org/10.1002/jmr.2419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4376589PMC
April 2015

Structural insights into the functional versatility of WW domain-containing oxidoreductase tumor suppressor.

Authors:
Amjad Farooq

Exp Biol Med (Maywood) 2015 Mar 7;240(3):361-74. Epub 2015 Feb 7.

Department of Biochemistry & Molecular Biology, Leonard Miller School of Medicine, University of Miami, Miami, FL 33136, USA

Recent work on WW domain-containing oxidoreductase (WWOX) tumor suppressor is beginning to shed new light on both the molecular mechanism of action of its WW domains as well as the contiguous catalytic domain. Herein, the structural basis underlying the ability of WW1 domain to bind to various physiological ligands and how the orphan WW2 tandem partner synergizes its ligand binding in the context of WW1-WW2 tandem module of WWOX is discussed. Notably, the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. In this manner, the association of WW2 domain with WW1 hinders ligand binding to the latter. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the fixed orientation of WW domains in the liganded conformation. Equally importantly, structure-guided functional approach suggests that the catalytic domain of WWOX likely serves as a retinal oxidoreductase that catalyzes the reversible oxidation and reduction of all-trans-retinal. Collectively, this review provides structural insights into the functional versatility of a key signaling protein with important implications on its biology.
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http://dx.doi.org/10.1177/1535370214561586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374002PMC
March 2015

Novel mutations confirm that COL11A2 is responsible for autosomal recessive non-syndromic hearing loss DFNB53.

Mol Genet Genomics 2015 Aug 30;290(4):1327-34. Epub 2015 Jan 30.

Laboratoire Procédés de Criblage Moléculaire et Cellulaire, Centre de Biotechnologie de Sfax, Université de Sfax, Route sidimansour Km 6, BP '1177', 3018, Sfax, Tunisia.

Hearing loss (HL) is a major public health issue. It is clinically and genetically heterogeneous.The identification of the causal mutation is important for early diagnosis, clinical follow-up, and genetic counseling. HL due to mutations in COL11A2, encoding collagen type XI alpha-2, can be non-syndromic autosomal-dominant or autosomal-recessive, and also syndromic as in Otospondylomegaepiphyseal Dysplasia, Stickler syndrome type III, and Weissenbacher-Zweymuller syndrome. However, thus far only one mutation co-segregating with autosomal recessive non-syndromic hearing loss (ARNSHL) in a single family has been reported. In this study, whole exome sequencing of two consanguineous families with ARNSHL from Tunisia and Turkey revealed two novel causative COL11A2 mutations, c.109G > T (p.Ala37Ser) and c.2662C > A (p.Pro888Thr). The variants identified co-segregated with deafness in both families. All homozygous individuals in those families had early onset profound hearing loss across all frequencies without syndromic findings. The variants are predicted to be damaging the protein function. The p.Pro888Thr mutation affects a -Gly-X-Y- triplet repeat motif. The novel p.Ala37Ser is the first missense mutation located in the NC4 domain of the COL11A2 protein. Structural model suggests that this mutation will likely obliterate, or at least partially compromise, the ability of NC4 domain to interact with its cognate ligands. In conclusion, we confirm that COL11A2 mutations cause ARNSHL and broaden the mutation spectrum that may shed new light on genotype-phenotype correlation for the associated phenotypes and clinical follow-up.
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http://dx.doi.org/10.1007/s00438-015-0995-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707654PMC
August 2015

A missense mutation in DCDC2 causes human recessive deafness DFNB66, likely by interfering with sensory hair cell and supporting cell cilia length regulation.

Hum Mol Genet 2015 May 18;24(9):2482-91. Epub 2015 Jan 18.

Laboratoire Procédés de Criblage Moléculaire et Cellulaire, Centre de Biotechnologie de Sfax, Université de Sfax, Sfax, Tunisie,

Hearing loss is the most common sensory deficit in humans. We show that a point mutation in DCDC2 (DCDC2a), a member of doublecortin domain-containing protein superfamily, causes non-syndromic recessive deafness DFNB66 in a Tunisian family. Using immunofluorescence on rat inner ear neuroepithelia, DCDC2a was found to localize to the kinocilia of sensory hair cells and the primary cilia of nonsensory supporting cells. DCDC2a fluorescence is distributed along the length of the kinocilium with increased density toward the tip. DCDC2a-GFP overexpression in non-polarized COS7 cells induces the formation of long microtubule-based cytosolic cables suggesting a role in microtubule formation and stabilization. Deafness mutant DCDC2a expression in hair cells and supporting cells causes cilium structural defects, such as cilium branching, and up to a 3-fold increase in length ratios. In zebrafish, the ortholog dcdc2b was found to be essential for hair cell development, survival and function. Our results reveal DCDC2a to be a deafness gene and a player in hair cell kinocilia and supporting cell primary cilia length regulation likely via its role in microtubule formation and stabilization.
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http://dx.doi.org/10.1093/hmg/ddv009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383862PMC
May 2015

Helix 8 and the i3 loop of the muscarinic M3 receptor are crucial sites for its regulation by the Gβ5-RGS7 complex.

Biochemistry 2015 Feb 20;54(4):1077-88. Epub 2015 Jan 20.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine , 1600 NW 10th Avenue, RMSB6024A, Miami, Florida 33136, United States.

The muscarinic M3 receptor (M3R) is a Gq-coupled receptor and is known to interact with many intracellular regulatory proteins. One of these molecules is Gβ5-RGS7, the permanently associated heterodimer of G protein β-subunit Gβ5 and RGS7, a regulator of G protein signaling. Gβ5-RGS7 can attenuate M3R-stimulated release of Ca(2+) from intracellular stores or enhance the influx of Ca(2+) across the plasma membrane. Here we show that deletion of amino acids 304-345 from the central portion of the i3 loop renders M3R insensitive to regulation by Gβ5-RGS7. In addition to the i3 loop, interaction of M3R with Gβ5-RGS7 requires helix 8. According to circular dichroism spectroscopy, the peptide corresponding to amino acids 548-567 in the C-terminus of M3R assumes an α-helical conformation. Substitution of Thr553 and Leu558 with Pro residues disrupts this α-helix and abolished binding to Gβ5-RGS7. Introduction of the double Pro substitution into full-length M3R (M3R(TP/LP)) prevents trafficking of the receptor to the cell surface. Using atropine or other antagonists as pharmacologic chaperones, we were able to increase the level of surface expression of the TP/LP mutant to levels comparable to that of wild-type M3R. However, M3R-stimulated calcium signaling is still severely compromised. These results show that the interaction of M3R with Gβ5-RGS7 requires helix 8 and the central portion of the i3 loop.
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http://dx.doi.org/10.1021/bi500980dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318586PMC
February 2015

Ligand binding to WW tandem domains of YAP2 transcriptional regulator is under negative cooperativity.

FEBS J 2014 Dec 4;281(24):5532-51. Epub 2014 Nov 4.

Department of Biochemistry and Molecular Biology, Leonard Miller School of Medicine, University of Miami, Miami, FL, USA.

YES-associated protein 2 (YAP2) transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of the WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules.
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http://dx.doi.org/10.1111/febs.13095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262544PMC
December 2014

Effect of osmolytes on the binding of EGR1 transcription factor to DNA.

Biopolymers 2015 Feb;103(2):74-87

Department of Biochemistry and Molecular Biology, Leonard Miller School of Medicine, University of Miami, Miami, FL, 33136.

Osmolytes play a key role in maintaining protein stability and mediating macromolecular interactions within the intracellular environment of the cell. Herein, we show that osmolytes such as glycerol, sucrose, and polyethylene glycol 400 (PEG400) mitigate the binding of early growth response (protein) 1 (EGR1) transcription factor to DNA in a differential manner. Thus, while physiological concentrations of glycerol only moderately reduce the binding affinity, addition of sucrose and PEG400 is concomitant with a loss in the binding affinity by an order of magnitude. This salient observation suggests that EGR1 is most likely subject to conformational equilibrium and that the osmolytes exert their effect via favorable interactions with the unliganded conformation. Consistent with this notion, our analysis reveals that while EGR1 displays rather high structural stability in complex with DNA, the unliganded conformation becomes significantly destabilized in solution. In particular, while liganded EGR1 adopts a well-defined arc-like architecture, the unliganded protein samples a comparatively large conformational space between two distinct states that periodically interconvert between an elongated rod-like shape and an arc-like conformation on a submicrosecond time scale. Consequently, the ability of osmolytes to favorably interact with the unliganded conformation so as to stabilize it could account for the negative effect of osmotic stress on EGR1-DNA interaction observed here. Taken together, our study sheds new light on the role of osmolytes in modulating a key protein-DNA interaction.
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http://dx.doi.org/10.1002/bip.22556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4244260PMC
February 2015

Reply to Teasdale and Collins: N-terminal region of FAM65B bears similarity to the PX-BAR module of SNX33.

Proc Natl Acad Sci U S A 2014 Sep 19;111(39):E4065. Epub 2014 Sep 19.

Dr. John T. Macdonald Foundation Department of Human Genetics, and John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136

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http://dx.doi.org/10.1073/pnas.1413860111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4191807PMC
September 2014

FAM65B is a membrane-associated protein of hair cell stereocilia required for hearing.

Proc Natl Acad Sci U S A 2014 Jul 23;111(27):9864-8. Epub 2014 Jun 23.

Dr. John T. Macdonald Foundation Department of Human Genetics,John P. Hussman Institute for Human Genomics, andDivision of Pediatric Genetics, Ankara University School of Medicine, Ankara 06100, Turkey

In a large consanguineous Turkish kindred with recessive nonsyndromic, prelingual, profound hearing loss, we identified in the gene FAM65B (MIM611410) a splice site mutation (c.102-1G>A) that perfectly cosegregates with the phenotype in the family. The mutation leads to exon skipping and deletion of 52-amino acid residues of a PX membrane localization domain. FAM65B is known to be involved in myotube formation and in regulation of cell adhesion, polarization, and migration. We show that wild-type Fam65b is expressed during embryonic and postnatal development stages in murine cochlea, and that the protein localizes to the plasma membranes of the stereocilia of inner and outer hair cells of the inner ear. The wild-type protein targets the plasma membrane, whereas the mutant protein accumulates in cytoplasmic inclusion bodies and does not reach the membrane. In zebrafish, knockdown of fam65b leads to significant reduction of numbers of saccular hair cells and neuromasts and to hearing loss. We conclude that FAM65B is a plasma membrane-associated protein of hair cell stereocilia that is essential for hearing.
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http://dx.doi.org/10.1073/pnas.1401950111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103326PMC
July 2014