Regulatory role of endoplasmic reticulum resident chaperone protein ERp29 in anti-murine ß-coronavirus host cell response.

Gap junctional intercellular communication (GJIC) involving astrocytes is important for proper CNS homeostasis. As determined in our previous studies, trafficking of the predominant astrocyte GJ protein, Connexin43 (Cx43), is disrupted in response to infection with a neurotropic murine ß-coronavirus (MHV-A59). However, how host factors are involved in Cx43 trafficking and the infection response is not clear. Here, we show that Cx43 retention due to MHV-A59 infection was associated with increased ER stress and reduced expression of chaperone protein ERp29. Treatment of MHV-A59-infected astrocytes with the chemical chaperone 4-sodium phenylbutyrate increased ERp29 expression, rescued Cx43 transport to the cell surface, increased GJIC, and reduced ER stress. We obtained similar results using an astrocytoma cell line (delayed brain tumor) upon MHV-A59 infection. Critically, delayed brain tumor cells transfected to express exogenous ERp29 were less susceptible to MHV-A59 infection and showed increased Cx43-mediated GJIC. Treatment with Cx43 mimetic peptides inhibited GJIC and increased viral susceptibility, demonstrating a role for intercellular communication in reducing MHV-A59 infectivity. Taken together, these results support a therapeutically targetable ERp29-dependent mechanism where ß-coronavirus infectivity is modulated by reducing ER stress and rescuing Cx43 trafficking and function.

To protect and modify double-stranded RNA - the critical roles of ADARs in development, immunity and oncogenesis.

Adenosine deaminases that act on RNA (ADARs) are present in all animals and function to both bind double-stranded RNA (dsRNA) and catalyze the deamination of adenosine (A) to inosine (I). As inosine is a biological mimic of guanosine, deamination by ADARs changes the genetic information in the RNA sequence and is commonly referred to as RNA editing. Millions of A-to-I editing events have been reported for metazoan transcriptomes, indicating that RNA editing is a widespread mechanism used to generate molecular and phenotypic diversity. Loss of ADARs results in lethality in mice and behavioral phenotypes in worm and fly model systems. Furthermore, alterations in RNA editing occur in over 35 human pathologies, including several neurological disorders, metabolic diseases, and cancers. In this review, a basic introduction to ADAR structure and target recognition will be provided before summarizing how ADARs affect the fate of cellular RNAs and how researchers are using this knowledge to engineer ADARs for personalized medicine. In addition, we will highlight the important roles of ADARs and RNA editing in innate immunity and cancer biology.

RNA binding by ADAR3 inhibits adenosine-to-inosine editing and promotes expression of immune response protein MAVS.

Members of the ADAR family of double-stranded RNA-binding proteins regulate one of the most abundant RNA modifications in humans, the deamination of adenosine to inosine. Several transcriptome-wide studies have been carried out to identify RNA targets of the active deaminases ADAR1 and ADAR2. However, our understanding of ADAR3, the brain-specific deaminase-deficient ADAR family member, is limited to a few transcripts. In this study, we identified over 3300 transcripts bound by ADAR3 and observed that binding of ADAR3 correlated with reduced editing of over 400 sites in the glioblastoma transcriptome. We further investigated the impact of ADAR3 on gene regulation of the transcript that encodes MAVS, an essential protein in the innate immune response pathway. We observed reduced editing in the MAVS 3' UTR in cells expressing increased ADAR3 or reduced ADAR1 suggesting ADAR3 acts as a negative regulator of ADAR1-mediated editing. While neither ADAR1 knockdown or ADAR3 overexpression affected MAVS mRNA expression, we demonstrate increased ADAR3 expression resulted in upregulation of MAVS protein expression. In addition, we created a novel genetic mutant of ADAR3 that exhibited enhanced RNA binding and MAVS upregulation compared with wildtype ADAR3. Interestingly, this ADAR3 mutant no longer repressed RNA editing, suggesting ADAR3 has a unique regulatory role beyond altering editing levels. Altogether, this study provides the first global view of ADAR3-bound RNAs in glioblastoma cells and identifies both a role for ADAR3 in repressing ADAR1-mediated editing and an RNA-binding dependent function of ADAR3 in regulating MAVS expression.

Borrelia burgdorferi Outer Surface Protein C Is Not the Sole Determinant of Dissemination in Mammals.

Lyme disease in the United States is most often caused by Borrelia burgdorferi sensu stricto. After a tick bite, the patient may develop erythema migrans at that site. If hematogenous dissemination occurs, the patient may then develop neurologic manifestations, carditis, or arthritis. Host-pathogen interactions include factors that contribute to hematogenous dissemination to other body sites. Outer surface protein C (OspC), a surface-exposed lipoprotein of B. burgdorferi, is essential during the early stages of mammalian infection. There is a high degree of genetic variation at the ospC locus, and certain ospC types are more frequently associated with hematogenous dissemination in patients, suggesting that OspC may be a major contributing factor to the clinical outcome of B. burgdorferi infection. In order to evaluate the role of OspC in B. burgdorferi dissemination, ospC was exchanged between B. burgdorferi isolates with different capacities to disseminate in laboratory mice, and these strains were then tested for their ability to disseminate in mice. The results indicated that the ability of B. burgdorferi to disseminate in mammalian hosts does not depend on OspC alone. The complete genome sequences of two closely related strains of B. burgdorferi with differing dissemination phenotypes were determined, but a specific genetic locus that could explain the differences in the phenotypes could not be definitively identified. The animal studies performed clearly demonstrated that OspC is not the sole determinant of dissemination. Future studies of the type described here with additional borrelial strains will hopefully clarify the genetic elements associated with hematogenous dissemination.

Cellulose ether treatment inhibits amyloid beta aggregation, neuroinflammation and cognitive deficits in transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is an incurable, progressive and devastating neurodegenerative disease. Pathogenesis of AD is associated with the aggregation and accumulation of amyloid beta (Aß), a major neurotoxic mediator that triggers neuroinflammation and memory impairment. Recently, we found that cellulose ether compounds (CEs) have beneficial effects against prion diseases by inhibiting protein misfolding and replication of prions, which share their replication mechanism with Aß. CEs are FDA-approved safe additives in foods and pharmaceuticals. Herein, for the first time we determined the therapeutic effects of the representative CE (TC-5RW) in AD using in vitro and in vivo models. Our in vitro studies showed that TC-5RW inhibits Aß aggregation, as well as neurotoxicity and immunoreactivity in Aß-exposed human and murine neuroblastoma cells. In in vivo studies, for the first time we observed that single and weekly TC-5RW administration, respectively, improved memory functions of transgenic 5XFAD mouse model of AD. We further demonstrate that TC-5RW treatment of 5XFAD mice significantly inhibited Aß oligomer and plaque burden and its associated neuroinflammation via regulating astrogliosis, microgliosis and proinflammatory mediator glial maturation factor beta (GMFß). Additionally, we determined that TC-5RW reduced lipopolysaccharide-induced activated gliosis and GMFß in vitro. In conclusion, our results demonstrate that CEs have therapeutic effects against Aß pathologies and cognitive impairments, and direct, potent anti-inflammatory activity to rescue neuroinflammation. Therefore, these FDA-approved compounds are effective candidates for developing therapeutics for AD and related neurodegenerative diseases associated with protein misfolding.

Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein.

Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.

Differentiation of reinfection from relapse in recurrent Lyme disease.

BACKGROUND: Erythema migrans is the most common manifestation of Lyme disease. Recurrences are not uncommon, and although they are usually attributed to reinfection rather than relapse of the original infection, this remains somewhat controversial. We used molecular typing of Borrelia burgdorferi isolates obtained from patients with culture-confirmed episodes of erythema migrans to distinguish between relapse and reinfection. METHODS: We determined the genotype of the gene encoding outer-surface protein C (ospC) of B. burgdorferi strains detected in cultures of skin or blood specimens obtained from patients with consecutive episodes of erythema migrans. After polymerase-chain-reaction amplification, ospC genotyping was performed by means of reverse line-blot analysis or DNA sequencing of the nearly full-length gene. Most strains were further analyzed by determining the genotype according to the 16S-23S ribosomal RNA intergenic spacer type, multilocus sequence typing, or both. Patients received standard courses of antibiotics for erythema migrans. RESULTS: B. burgdorferi isolates obtained from 17 patients who received a diagnosis of erythema migrans between 1991 and 2011 and who had 22 paired episodes of this lesion (initial and second episodes) were available for testing. The ospC genotype was found to be different at each initial and second episode. Apparently identical genotypes were identified on more than one occasion in only one patient, at the first and third episodes, 5 years apart, but different genotypes were identified at the second and fourth episodes. CONCLUSIONS: None of the 22 paired consecutive episodes of erythema migrans were associated with the same strain of B. burgdorferi on culture. Our data show that repeat episodes of erythema migrans in appropriately treated patients were due to reinfection and not relapse. (Funded by the National Institutes of Health and the William and Sylvia Silberstein Foundation.).

Menstrual characteristics of adolescent athletes: a study from West Bengal, India.

The present study investigates: (i) differences in menstrual characteristics of athlete and non-athlete adolescents; (ii) relationship between menstrual characteristics, anthropometric variables, athletic status and socioeconomic status. The present study was conducted among 159 unmarried adolescents (80 non-athletes and 79 athletes) of age 15 to 19 years. The study participants belong to Bengali speaking Hindu ethnic group of Kolkata, the capital city of West Bengal State of India. Data were collected on socio-demographic and menstrual characteristics using pre-tested questionnaires. Anthropometric measurements were taken following standard methods. Descriptive statistics were used to understand the differences in menstrual characteristics between athletes and non-athletes, stepwise linear regression analyses were carried out to predict age at menarche, menstrual cycle length and duration of menstrual discharge using socio-demographic and anthropometric variables as well as athletic status as independent variables. Logistic (binary) regression was carried out to assess the strength of association between menstrual characteristics (as dependent variables) and athletic status, socio-demographic and anthropometric variables, and other menstrual characteristics (independent variables). The study participants differ significantly (p < or = 0.05) for certain menstrual characteristics such as age at menarche, cycle length, skipped cycle, premenstrual syndrome, heavy discharge when compared for their athletic status. Certain anthropometric and socioeconomic variables were found to be significantly associated with their menstrual characteristics. The study results demonstrate that menstrual functioning among adolescents is significantly influenced by their athletic status. The findings of this study would help health care professionals to devise future health care programs for adolescents in gen- eral and athletes in particular.

A comparative study in left-sided breast cancer treated with moderate deep inspiratory breath hold versus free breathing.

BACKGROUND: The moderate deep inspiratory breath hold (mDIBH) is a modality famed for cardiac sparing. Prospective studies based on this are few from the eastern part of the world and India. We intend to compare the dosimetry between mDIBH and free-breathing (FB) plans. METHODS: Thirty-two locally advanced left breast cancer patients were taken up for the study. All patients received a dose of 50 Gy in 25 fractions to the chest wall/intact breast, followed by a 10-Gy boost to the lumpectomy cavity in the case of breast conservation surgery. All the patients were treated in mDIBH using active breath coordinator (ABC). The data from the two dose volume histograms were compared regarding plan quality and the doses received by the organs at risk. Paired t-test was used for data analysis. RESULTS: The dose received by the heart in terms of V5, V10, and V30 (4.55% vs 8.39%) and mean dose (4.73 Gy vs 6.74 Gy) were statistically significant in the ABC group than that in the FB group (all p-values < 0.001). Also, the dose received by the LADA in terms of V30 (19.32% vs 24.87%) and mean dose (32.99 Gy vs 46.65 Gy) were significantly less in the ABC group. The mean treatment time for the ABC group was 20 min, while that for the free-breathing group was 10 min. CONCLUSIONS: Incorporating ABC-mDIBH for left-sided breast cancer radiotherapy significantly reduces the doses received by the heart, LADA, and left and right lung, with no compromise in plan quality but with an increase in treatment time.

Detection of Borrelia burgdorferi nucleic acids after antibiotic treatment does not confirm viability.

The persistence of dormant, noncultivable Borrelia burgdorferi after ceftriaxone treatment was examined. B. burgdorferi isolates were cultivated in Barbour-Stoenner-Kelly medium in the presence or absence of ceftriaxone, and cultures were monitored for up to 56 days. Viability of B. burgdorferi was assessed by subculture, growth, morphology, and pH (as a surrogate for metabolic activity). In addition, the presence of B. burgdorferi DNA and mRNA was assayed by PCR and by real-time reverse transcription (RT)-PCR, respectively. Spirochetes could not be successfully subcultured by day 3 after exposure to ceftriaxone. In cultures treated with ceftriaxone, the pH of the culture medium did not change through day 56, whereas it declined by at least 1 pH unit by 14 days in untreated cultures. These results suggest that B. burgdorferi viability is rapidly eliminated after antibiotic treatment. Nevertheless, DNA was detected by B. burgdorferi-specific PCR for up to 56 days in aliquots from both ceftriaxone-treated and untreated cultures. In addition, although ceftriaxone treatment resulted in a reduction in the quantities of transcript for ospC, ospA, flaB, and pfk, certain mRNAs could be detected through day 14. Transcript for all 4 genes was essentially undetectable after 28 days of treatment. Taken together, the results suggest that B. burgdorferi DNA and mRNA can be detected in samples long after spirochetes are no longer viable as assessed by classic microbiological parameters. PCR positivity in the absence of culture positivity following antibiotic treatment in animal and human studies should be interpreted with caution.

Metastatic Renal Cell Carcinoma Masquerading as a Laryngeal Tumor: A Case Report.

Renal cell carcinoma (RCC) commonly metastasizes to various organs such as the lungs, liver, bones, and brain. However, isolated metastases to the head and neck region, especially the larynx, are very rare. This report presents a case of laryngeal growth that was eventually confirmed to be a metastatic deposit from an undiagnosed RCC. We report a case of a 66-year-old male who presented to the clinic with painless neck swelling and a change in voice. The scan showed a soft tissue mass in the thyroid cartilage. Histopathology of the resected laryngeal tumor confirmed metastatic clear cell carcinoma. A metastatic workup revealed a renal mass, and the patient underwent laparoscopic adrenal-sparing left cytoreductive nephrectomy. The histopathological examination established the diagnosis of clear cell RCC. Subsequently, the patient was treated with pembrolizumab and lenvatinib. Follow-up imaging showed no residual or recurrent lesions. This case highlights the rarity of laryngeal metastasis from RCC and the importance of an accurate diagnosis through advanced imaging and histopathological examination.

Genetic Variations and Altered Blood mRNA Level of Circadian Genes and BDNF as Risk Factors of Post-Stroke Cognitive Impairment Among Eastern Indians.

Post-stroke cognitive impairment (PSCI) is a clinical outcome in around 30% of post-stroke survivors. BDNF is a major gene in this regard. It is regulated by circadian rhythm. The circadian genes are correlated with stroke timings at molecular level. However, studies suggesting the role of these on susceptibility to PSCI are limited. We aim here to determine: (a) genetic risk variants in circadian clock genes, BDNF and (b) dysregulation in expression level of CLOCK, BMAL1, and BDNF that may be associated with PSCI. BDNF (rs6265G/A, rs56164415C/T), CLOCK (rs1801260T/C, rs4580704G/C), and CRY2 (rs2292912C/G) genes variants were genotyped among 119 post-stroke survivors and 292 controls from Eastern part of India. In addition, we analyzed their gene expression in Peripheral blood Mononuclear cells (PBMC) from 15 PSCI cases and 12 controls. The mRNA data for BDNF was further validated by its plasma level through ELISA (n = 38). Among the studied variants, only rs4580704/CLOCK showed an overall association with PSCI (P = 0.001) and lower Bengali Mini-Mental State Examination (BMSE) score. Its 'C' allele showed a correlation with attention deficiency. The language and memory impairments showed association with rs6265/BDNF, while the 'CC' genotype of rs2292912/CRY2 negatively influenced language and executive function. A significant decrease in gene expression for CLOCK and BDNF in PBMC (influenced by specific genotypes) of PSCI patients was observed than controls. Unlike Pro-BDNF, plasma-level mBDNF was also lower in them. Our results suggest the genetic variants in CLOCK, CRY2, and BDNF as risk factors for PSCI among eastern Indians. At the same time, a lowering expression of CLOCK and BDNF genes in PSCI patients than controls describes their transcriptional dysregulation as underlying mechanism for post-stroke cognitive decline.

Overexpression of facilitative glucose transporter-3 and membrane procoagulation in maternal platelets of preeclamptic pregnancy.

BACKGROUND: Preeclampsia (PE) is a hypertensive disorder during pregnancy that results in significant adverse maternal and neonatal outcomes. Platelet activation is present in PE and contributes to the thrombo-hemorrhagic states of the disorder. However, the mechanisms that initiate and/or sustain platelet activation in PE are ill-defined. OBJECTIVES: We aimed to characterise this mechanism and the procoagulant potentials of platelets in PE. METHODS: In this quantitative observational study, we analyzed platelet procoagulant membrane dynamics in patients with PE (n = 21) compared with age-matched normotensive pregnancies (n = 20), gestational hypertension (n = 10), and non-pregnant female controls (n = 19). We analyzed fluorescently labeled indicators of platelet activation, bioenergetics, and procoagulation (phosphatidylserine exposure and thrombin generation), coupled with high-resolution imaging and thrombelastography. We then validated our findings using flow cytometry, immunoassays, classical pharmacology, and convolutional neural network analysis. RESULTS: PE platelets showed significant ultra-structural remodeling, are more extensively preactivated than in healthy pregnancies and can circulate as microaggregates. Preactivated platelets of PE externalized phosphatidylserine and thrombin formed on the platelet membranes. Platelets' expression of facilitative glucose transporter-1 increased in all pregnant groups. However, PE platelets additionally overexpress glucose transporter-3 to enhance glucose uptake and sustain activation and secretion events. Although preeclampsia platelets exposed to subendothelial collagen showed incremental activation, the absolute hemostatic response to collagen was diminished, and likely contributed to greater blood loss perioperatively. CONCLUSIONS: We revealed 2 bioenergetic mediators in the mechanism of sustained platelet procoagulation in preeclampsia. Although glucose transporter-1 and glucose transporter-3 remain elusive antiprocoagulant targets, they may be sensitive monitors of PE onset and progression.

Kupffer cell-like syncytia replenish resident macrophage function in the fibrotic liver.

Kupffer cells (KCs) are localized in liver sinusoids but extend pseudopods to parenchymal cells to maintain their identity and serve as the body's central bacterial filter. Liver cirrhosis drastically alters vascular architecture, but how KCs adapt is unclear. We used a mouse model of liver fibrosis and human tissue to examine immune adaptation. Fibrosis forced KCs to lose contact with parenchymal cells, down-regulating "KC identity," which rendered them incapable of clearing bacteria. Commensals stimulated the recruitment of monocytes through CD44 to a spatially distinct vascular compartment. There, recruited monocytes formed large aggregates of multinucleated cells (syncytia) that expressed phenotypical KC markers and displayed enhanced bacterial capture ability. Syncytia formed via CD36 and were observed in human cirrhosis as a possible antimicrobial defense that evolved with fibrosis.

Tracking Zone-wise perturbation during unfolding of some globular proteins using Eu(III) complex of Tetracycline as a probe exhibiting Stark splitting.

Enhanced 'Antenna effect' of a suitably designed ternary complex of Eu(III), Tetracycline hydrochloride (TC) and globular proteins viz bovine serum albumin (BSA), human serum albumin (HSA) and ß-lactoglobulin A (BLGA) in aqueous medium is employed to characterize the different partially unfolded states along with investigation of the micro- heterogeneous environment of the proteins during their stepwise unfolding. The zone-wise perturbation for the proteins upon denaturation by Urea and Guanidine hydrochloride (Gdn. HCl) is followed by the emission of Eu(III) through 'Antenna Effect' and that of the tryptophan (Trp) residues of the proteins as a function of denaturants both by steady state and time resolved emission study. With Gdn. HCl as denaturant, both BSA and BLGA show quenching of Eu(III) emission compared to pure protein while HSA exhibits an enhancement of antenna effect during unfolding as compared to that in its absence. In the presence of Urea, HSA and BSA show enhancement of antenna effect accompanied by Stark splitting of the 5D0→7F2 transition of Eu(III) although BLGA follows the similar pattern of quenching of Eu(III) emission as observed with Gdn. HCl without any Stark splitting. The proteins exhibit a two state transition with ΔGD values of ~ 2-3 kcal mol-1. Thus the use of Eu(III) emission as an efficient probe is advocating here to rationalize the microenvironment of the proteins during their stepwise unfolding.

Caveolin-initiated macropinocytosis is required for efficient silica nanoparticles' transcytosis across the alveolar epithelial barrier.

Removal of particulate materials that would otherwise cumulate within the airspace and hinder the gas exchange is one of the central processes of maintaining lung homeostasis. While the importance of the particle uptake by alveolar macrophages and their expulsion via the airways mucociliary escalator is well established, very little is known about the alternative route for removing the particles via direct crossing the lung epithelium for transfer into the pulmonary lymph and bloodstream. This study dissected sequential mechanisms involved in nanoparticle transcytosis through the alveolar epithelial cell layer. By a combination of live cell, super resolution, and electron microscopy and RNA interference study, we have dissected temporal steps of nanoparticle transcytosis through alveolar epithelium. Our study revealed that caveolin is essential for the firm adhesion of the silica nanoparticle agglomerates to the apical membrane and their subsequent rapid internalization with the help of macropinocytic elements C-terminal-binding protein1 and Rabankyrin-5 but not dynamin. Actin, but not microtubules, played a major role in nanoparticle uptake and subsequent transportation. The compartments with nanoparticles were tethered to trans-Golgi network to be jointly transported along actin stress fibers across the cytoplasm, employing a myosin-dependent mechanism. The trans-Golgi nanoparticle transport machinery was positive to Rab6A, a marker linked to vesicle exocytosis. Exocytosis was primarily occurring at the basolateral plane of the alveolar epithelial cells. The high-proficiency novel caveolin and Rabankyrin-5 associated uptake and transcellular transport of nanoparticles across the AEC barrier supports its importance in clearance of amorphous silica and other types of non-inflammatory nanoparticles that are rapidly removed from the lungs following their inhalation.

RNA immunoprecipitation to identify in vivo targets of RNA editing and modifying enzymes.

The past decade has seen an exponential increase in the identification of individual nucleobases that undergo base conversion and/or modification in transcriptomes. While the enzymes that catalyze these types of changes have been identified, the global interactome of these modifiers is still largely unknown. Furthermore, in some instances, redundancy among a family of enzymes leads to an inability to pinpoint the protein responsible for modifying a given transcript merely from high-throughput sequencing data. This chapter focuses on a method for global identification of transcripts recognized by an RNA modification/editing enzyme via capture of the RNAs that are bound in vivo, a method referred as RNA immunoprecipitation (RIP). We provide a guide of the major issues to consider when designing a RIP experiment, a detailed experimental protocol as well as troubleshooting advice. The RIP protocol presented here can be readily applied to any organism or cell line of interest as well as both RNA modification enzymes and RNA-binding proteins (RBPs) that regulate RNA modification levels. As mentioned at the end of the protocol, the RIP assay can be coupled to high-throughput sequencing to globally identify bound targets. For more quantitative investigations, such as how binding of an RNA modification enzyme/regulator to a given target changes during development/in specific tissues or assessing how the presence or absence of RNA modification affects transcript recognition by a particular RBP (irrespective of a role for the RBP in modulating modification levels); the RIP assay should be coupled to quantitative real-time PCR (qRT-PCR).

Small Airways, Big Problem: Extrafine beclomethasone/formoterol in asthma and chronic obstructive pulmonary disease.

Asthma and chronic obstructive pulmonary disease (COPD) are common chronic respiratory diseases characterized by an inflammatory process that extends from the central to peripheral airways. Conventional pressurized metered-dose inhalers and most dry-powder inhalers emit drug particles too large to target the small airways effectively. Advancements in drug formulation have given rise to a new generation of inhalers that can generate aerosols with extrafine drug particles that leads to more effective aerosol penetration into the lung periphery. An extrafine formulation of inhaled beclomethasone/formoterol (BDP-FF) with enhanced lung deposition is now available. This document reviews the various real-world and controlled studies that have evaluated the efficacy of extrafine BDP-FF in asthma and COPD.

Seroprevalence of selected viral pathogens in pigs reared in organized farms of Meghalaya from 2014 to 16.

AIM: A pilot study was carried out to find out the seroprevalence of Porcine circovirus 2 (PCV2), classical swine fever virus (CSFV), and Porcine respiratory and reproductive syndrome virus (PRRS) in pig population of Meghalaya. MATERIALS AND METHODS: Serum samples were collected from piglets of 40-45 days age group, growers, and sows reared under organized and unorganized management in 11 districts of Meghalaya situated in the Khasi, Jaintia, and Garo hills divisions in the time period of 2014-2016 from apparently healthy and suspected pigs. Seroprevalence of PCV2, CSFV, and PRRS specific antibodies was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: A total of 1899 serum samples were collected and screened using antibody ELISA kits specific for PCV2, CSFV, and PRRS. The highest antibody prevalence during the selected time periods was detected for PCV2 (80.8% in 2014, 79.1% in 2015, and 96.2% in 2016) followed by CSFV (76.4% in 2014, 66.09% in 2015, and 25.5% in 2016) and PRRS (2.8% in 2014, 2.7% in 2015, and 3.62% in 2016). The result indicates high seroprevalence for PCV2, which can be considered as an inducement factor due to the immunosuppressive nature of the virus, for animals being susceptible to other pathogens in farms where airborne transmission of PCV2 and postweaning multisystemic wasting syndrome among animals reared in close pens can be a major possibility. CONCLUSIONS: The data from this study indicates ubiquitous prevalence of PCV2 antibodies in the farm animals along with the endemic presence of swine fever and emergence of PRRS in an organized farm. There are few reports regarding PCV2 infections/outbreaks in pigs associated with reproductive failure from northern and southern part of India, but till date, there are no reports regarding concomitant infection of CSFV and PCV2 from India. Considerable high seropositivity of PCV2 indicates the need for high impact hygiene practice in farms, routine seromonitoring and implementation the vaccination program. To the author's best knowledge, this is the first documented report on the seroprevalence of PCV2, CSFV, and PRRS from pig population of Meghalaya.

Porcine circovirus 2 in the North Eastern region of India: Disease prevalence and genetic variation among the isolates from areas of intensive pig rearing.

Porcine Circovirus type-2 (PCV-2) is considered as a major threat to the piggery sector in India. To ascertain the epidemiological status and infection level of PCV2, a pilot study was undertaken to find out the prevalence of PCV2 in swine population by ELISA and PCR in the interior and border areas of Meghalaya which includes the area where accessibility and medical aid is a rare phenomenon. A total of 249 serum samples were collected from October 2014 to February 2016 from three divisions of Meghalaya: Khasi, Jaintia and Garo Hills Divisions. The mean positivity of PCV-2 antibodies in suspected sera was 83.93% whereas 62.25% of the suspected samples respectively were found to contain PCV2 as detected by PCR. Additional 190 tissue samples were collected during necropsy from both symptomatic and asymptomatic animals following reported outbreak in this region, which indicated a mean positivity of 18.94% (36/190); out of which 13 samples were subjected to sequencing to find out the genetic diversity of PCV2 amongst the field isolates. Molecular characterization and phylogenetic analysis of PCV2 isolates based on cap gene depicted genetic diversity among the strains in pig population of Meghalaya as the isolates belonged to PCV2a, PCV2b-1c and PCV2d genotypes; identification of the PCV2d genotype is probably the first report from Meghalaya. Four isolates forming an outlier group in the phylogenetic tree were arising out of natural inter-genotypic recombination between PCV2a and PCV2b. PCV2 being immunosuppressive in nature impairs the host immune response increasing the susceptibility to other co-infections leading to disease severity and high mortality in pig population. This baseline data gives a brief epidemiological status of PCV2 infection and circulating PCV2 genotype in this region which will be useful in the formulation of control and eradication programs in remotes areas of Meghalaya where accessibility is less and vaccination is a rare practice.