Trade Policy Reforms in Pakistan

This paper examines the trade policy reforms in both import and export sectors of Pakistan and provides a critical analysis of the measures undertaken during various regimes to liberalize the economy. The paper discusses the relevant theoretical and empirical research and attempts to evaluate the existing tariff structure under various trade liberalization regimes and substantiate, with the statistics in view of the trade theory, relevant reports and research papers. Alongside it also discusses the tax reforms relevant to trade policy, while briefly discussing key concerns and challenges faced by the policy makers in both formulation and implementation of tariff reforms, such as, lobbying of special interest groups, government revenue constraints and illegal trade across the borders. Finally, it gives an account of export sector and discusses reasons for its weak performance despite government interventions

An Insight Into the Functional Role of Thioredoxin Reductase, a Selenoprotein, in Maintaining Normal Native Microbiota in the Gult Coast tick (\u3ci\u3eAmbylomma maculatum\u3c/i\u3e)

Tick selenoproteins have been associated with antioxidant activity in ticks. Thioredoxin reductase (TrxR), also a selenoprotein, belongs to the pyridine nucleotide‐disulphide oxidoreductase family of proteins and is an important antioxidant. Molecular interactions between native microbiota and tick hosts have barely been investigated to date. In this study, we determined the functional role of TrxR in tick feeding and in maintenance of the native microbial community. TrxR transcript levels remained high and microbial load was reduced throughout tick attachment to the vertebrate host. RNA interference (RNAi) showed that depletion of TrxR activity did not interfere with tick haematophagy or phenotype but did reduce the viability of the microbiome within the tick tissues, presumably by perturbing redox homeostasis. The transcriptional activity of various antioxidant genes remained unaffected whereas the antioxidant genes Manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (Cu/Zn SOD) and selenoprotein M (SelM) were significantly down‐regulated in salivary glands of the ticks subjected to RNAi. The perturbed TrxR enzymatic activity in the knocked‐down tick tissues negatively affected the bacterial load as well. Furthermore, we observed the altered bacterial profiles in TrxR‐silenced tick tissues. Taken together, these results indicate an essential functional role for TrxR in maintaining the bacterial community associated with ticks

An Insight into the Sialome of the Lone Star Tick, \u3ci\u3eAmbylomma americanum\u3c/i\u3e, With a Glimpse On Its Time Dependent Gene Expression

Hard ticks feed for several days or weeks on their hosts. Blood feeding is assisted by tick saliva, which is injected in the host skin regularly, alternating with blood ingestion. Tick saliva contains hundreds or thousands of different peptides and other bioactive compounds that assist feeding by inhibiting their hosts’ blood clotting, platelet aggregation, vasoconstriction, as well as pain and itching. Immunomodulatory and antimicrobial peptides are also found in tick saliva. Molecular characterization of tick salivary compounds, or its sialome (from the Greek sialos = saliva), helps identification of possible antigens that might confer anti-tick immunity, as well as identifying novel pharmacologically active compounds. Amblyomma americanum is a major nuisance tick in Eastern and Southern US, being a vector of Theileria and Ehrlichia bacteria to animals and humans. Presently we report an RNAseq study concerning the salivary glands of adult female A. americanum ticks, which involved sequencing of four libraries collected at different times of feeding. A total of 5,792 coding sequences were deduced from the transcriptome assembly, 3,139 of which were publicly deposited, expanding from the previously available 146 salivary sequences found in GenBank. A remarkable time-dependent transcript expression was found, mostly related to secretory products, supporting the idea that ticks may have several “sialomes” that are expressed at different times during feeding. The molecular nature of this sialome switching remains unknown. The hyperlinked spreadsheet containing the deduced coding sequences can be found at http://exon.niaid.nih.gov/transcriptome/Amb_americanum/Ambame-web.xlsx

Tick Saliva and the Alpha-Gal Syndrome: Finding a Needle in a Haystack

Ticks and tick-borne diseases are significant public health concerns. Bioactive molecules in tick saliva facilitate prolonged blood-feeding and transmission of tick-borne pathogens to the vertebrate host. Alpha-gal syndrome (AGS), a newly reported food allergy, is believed to be induced by saliva proteins decorated with a sugar molecule, the oligosaccharide galactose-⍺-1,3-galactose (α-gal). This syndrome is characterized by an IgE antibody-directed hypersensitivity against α-gal. The α-gal antigen was discovered in the salivary glands and saliva of various tick species including, the Lone Star tick (Amblyomma americanum). The underlying immune mechanisms linking tick bites with α-gal-specific IgE production are poorly understood and are crucial to identify and establish novel treatments for this disease. This article reviews the current understanding of AGS and its involvement with tick species

Temporal Gene Expression Analysis and RNA Silencing of Single and Multiple Members of Gene Family in the Lone Star Tick \u3ci\u3eAmblyomma americanum\u3c/i\u3e

Saliva is an integral factor in the feeding success of veterinary and medically important ticks. Therefore, the characterization of the proteins present in tick saliva is an important area of tick research. Here, we confirmed previously generated sialotranscriptome data using quantitative real-time PCR. The information obtained in this in-depth study of gene expression was used to measure the effects of metalloprotease gene silencing on tick feeding. We analyzed the temporal expression of seven housekeeping genes and 44 differentially expressed salivary molecules selected from a previously published Amblyomma americanum sialotranscriptome. Separate reference genes were selected for the salivary glands and midgut from among the seven housekeeping genes, to normalize the transcriptional expression of differentially expressed genes. The salivary gland reference gene, ubiquitin, was used to normalize the expression of 44 salivary genes. Unsurprisingly, each gene family was expressed throughout the blood meal, but the expression of specific genes differed at each time point. To further clarify the complex nature of the many proteins found in the saliva, we disrupted the translation of several members of the metalloprotease family. Intriguingly, the nucleotide sequence similarity of the reprolysin metalloprotease gene family is so homologous that a single synthesized dsRNA sequence knocked down multiple members of the family. The use of multigene knockdown yielded a more significant picture of the role of metalloproteases in tick feeding success, and changes were observed in the female engorgement weight and larval hatching success. Interestingly, the depletion of metalloprotease transcripts also reduced the total number of bacteria present in the salivary glands. These data provide insight into the expression and functions of tick salivary proteins expressed while feeding on its host

Catalase is a Determinant of the Colonization and Transovarial Transmission of \u3ci\u3eRickettsia parkeri\u3c/i\u3e in the Gulf Coast Tick \u3ci\u3eAmblyomma maculatum\u3c/i\u3e

The Gulf Coast tick (Amblyomma maculatum) has evolved as a competent vector of the spotted‐fever group rickettsia, Rickettsia parkeri. In this study, the functional role of catalase, an enzyme responsible for the degradation of toxic hydrogen peroxide, in the colonization of the tick vector by R. parkeri and transovarial transmission of this pathogen to the next tick generation, was investigated. Catalase gene (CAT) expression in midgut, salivary glands and ovarian tissues exhibited a 2–11‐fold increase in transcription level upon R. parkeri infection. Depletion of CAT transcripts using an RNA‐interference approach significantly reduced R. parkeri infection levels in midgut and salivary gland tissues by 53–63%. The role of CAT in transovarial transmission of R. parkeri was confirmed by simultaneously blocking the transcript and the enzyme by injecting double‐stranded RNA for CAT and a catalase inhibitor (3‐amino‐1,2,4‐triazole) into gravid females. Simultaneous inhibition of the CAT transcript and the enzyme significantly reduced the egg conversion ratio with a 44% reduction of R. parkeri transovarial transmission. These data suggest that catalase is required for rickettsial colonization of the tick vector and transovarial transmission to the next generation

\u3ci\u3eAmblyomma maculatum\u3c/i\u3e SECIS Binding Protein 2 and Putative Selenoprotein P are Indispensable for Pathogen Replication and Tick Fecundity

Selenium, a vital trace element, is incorporated into selenoproteins to produce selenocysteine. Our previous studies have revealed an adaptive co-evolutionary process that has enabled the spotted fever-causing tick-borne pathogen Rickettsia parkeri to survive by manipulating an antioxidant defense system associated with selenium, which includes a full set of selenoproteins and other antioxidants in ticks. Here, we conducted a systemic investigation of SECIS binding protein 2 (SBP2) and putative selenoprotein P (SELENOP) by transcript silencing in adult female Gulf-coast ticks (Amblyomma maculatum). Knockdown of the SBP2 and SELENOP genes depleted the respective transcript levels of these tick selenogenes, and caused differential regulation of other antioxidants. Importantly, the selenium level in the immature and mature tick stages increased significantly after a blood meal, but the selenium level decreased in ticks after the SBP2 and SELENOP knockdowns. Moreover, the SBP2 knockdown significantly impaired both transovarial transmission of R. parkeri to tick eggs and egg hatching. Overall, our data offer new insight into the relationship between the SBP2 selenoprotein synthesis gene and the putative tick SELENOP gene. It also augments our understanding of selenoprotein synthesis, selenium maintenance and utilization, and bacterial colonization of a tick vector

\u3ci\u3eRickettsia parkeri\u3c/i\u3e Colonization in \u3ci\u3eAmbylomma maculatum\u3c/i\u3e: The Role of Superoxide Dismutases

Background The Gulf Coast tick (Amblyomma maculatum) is an arthropod vector of Rickettsia parkeri, the causative agent of American boutonneuse fever and an infectious agent of public health significance. In this study, we evaluated the biological significance of the superoxide dismutases (SODs) of A. maculatum in hematophagy and R. parkeri colonization within the tick host. Methods An RNA interference approach was used to measure the functional roles of tick SODs (Cu/Zn-SOD and Mn-SOD) in R. parkeri colonization of the tick vector. Total microbial load, R. parkeri infection rate, and compensatory mechanisms by tick genes were examined using quantitative polymerase chain reaction (PCR) and quantitative reverse-transcriptase PCR assays. SOD enzymatic activity assays and malondialdehyde (MDA) lipid peroxidation were employed to determine the redox states in the tick tissues. Results Knockdown of the Cu/Zn-SOD gene caused the upregulation of Mn-SOD in transcript levels. Single and dual knockdowns of the SOD genes caused an increase in MDA lipid peroxidation while SOD enzymatic activities did not show a significant change. Mn-SOD knockdown resulted in a substantial increase in the microbial load; however, Cu/Zn-SOD transcript depletion prompted an upsurge in the midgut bacterial load, and significantly decreased the bacterial load in salivary gland tissues. Additionally, Cu/Zn-SOD transcript silencing led to significantly fewer R. parkeri DNA copy numbers in both tick tissues (midguts and salivary glands). Conclusions SOD enzymes play an important function in the regulation of bacterial communities associated with tick vectors and also in the defense mechanisms against the damage caused by reactive oxygen species within the tick. Knockdown experiments increased the levels of total oxidative stress in ticks, revealing the interplay between SOD isozymes that results in the transcriptional regulation of tick antioxidants. Moreover, the tick\u27s Cu/Zn-SOD aids in the colonization of R. parkeri in tick tissues providing evidence of A. maculatum\u27s vectorial success for a spotted fever group rickettsial pathogen

Functional Genomics Tool: Gene Silencing in \u3ci\u3eIxodes scapularis\u3c/i\u3e Eggs and Nymphs by Electroporated dsRNA

Background Ticks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation. Results The genes encoding the putative Phospholipase A2 (PLA2), cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of β-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA2, cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs. Conclusions Our study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks