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Objective The significance of the presence of microorganisms and polymorphonuclear cells in the tracheal aspirates (TAs) of ventilated preterm infants is not well known. Our aim was to correlate information about the presence of polymorphonuclear cells with microbial growth and the cytokine milieu in the TAs of infants who have been intubated for >7 days. Study Design TAs were collected from infants who had been intubated for 7 days or longer. Respiratory cultures were performed, and infants were stratified based on the presence and abundance of polymorphonuclear cells and microbial growth. Cytokines were measured in the TAs of each of the respective groups. Results In the 19 infants whose TAs were collected, the presence of at least moderate WBC with presence of microbial growth was positively associated with the presence of interleukin (IL)-10, IL-1β, IL-8, and tumor necrosis factor (TNF)-α. The presence of at least moderate WBC, with or without microbial growth, was correlated positively with the presence of IL-8 and TNF-α. Conclusion There are higher levels of proinflammatory cytokines (especially, IL-10, IL-1β, and TNF-α) in TAs with higher cell counts and presence of microbial growth. The findings suggest that the presence of microbial growth correlated with inflammatory burden and warrant a larger study to see if treatment of microbial growth can ameliorate the inflammatory burden. Key Points

Preterm infants with bronchopulmonary dysplasia (BPD), characterized by pulmonary inflammation leading to impaired alveolarization and vascular dysregulation, have an increased risk of abnormal lung function in infancy, childhood, and adulthood. These include a heightened risk of pulmonary hypertension, and respiratory illnesses. MicroRNAs (miRNAs) are known to disrupt normal lung development and function by interrupting alveolarization and vascularization resulting in the development of BPD. Among the various miRs involved in BPD, miR34a has been shown to have a significant role in BPD pathogenesis. Targeting miR34a or its downstream targets may be a promising therapeutic intervention for BPD. In this review, we summarize the data on cellular arrest, proliferation, differentiation, epithelial-mesenchymal transition, mitochondrial dysfunction, and apoptosis impacted by miR34a in the development of BPD pulmonary phenotypes while predicting the future perspective of miR34a in BPD.

Background Ion channels are a large family of transmembrane proteins, accessible by soluble membrane-impermeable molecules, and thus are targets for development of therapeutic drugs. Ion channels are the second most common target for existing drugs, after G protein-coupled receptors, and are expected to make a big impact on precision medicine in many different diseases including wound repair and regeneration. Research has shown that endogenous bioelectric signaling mediated by ion channels is critical in non-mammalian limb regeneration. However, the role of ion channels in regeneration of limbs in mammalian systems is not yet defined. Methods To explore the role of potassium channels in limb wound repair and regeneration, the hindlimbs of mouse embryos were amputated at E12.5 when the wound is expected to regenerate and E15.5 when the wound is not expected to regenerate, and gene expression of potassium channels was studied. Results Most of the potassium channels were downregulated, except for the potassium channel kcnj8 (Kir6.1) which was upregulated in E12.5 embryos after amputation. Conclusion This study provides a new mouse limb regeneration model and demonstrates that potassium channels are potential drug targets for limb wound healing and regeneration.

Bronchopulmonary dysplasia (BPD) is a disease prevalent in preterm babies with a need for supplemental oxygen, resulting in impaired lung development and dysregulated vascularization. Epidemiologic studies have shown that males are more prone to BPD and have a delayed recovery compared with females, for reasons unknown. Herein, we tried to recapitulate mild, moderate, and severe BPD, using two different strains of mice, in males and females: CD1 (outbred) and C57BL/6 (inbred). Aside from higher body weight in the CD1 strain, there were no other gross morphologic differences with respect to alveolar development between the two strains. With respect to lung morphology after oxygen exposure, females had less injury with better preservation of alveolar chord length and decreased alveolar protein leak and inflammatory cells in the bronchoalveolar lavage fluid. In addition, housekeeping genes, which are routinely used as loading controls, were expressed differently in males and females. In the BPD mouse model, gonadotropin-releasing hormone was increased in females compared with males. Specific miRNAs (miR-146 and miR-34a) were expressed differently in the sexes. In the severe BPD mouse model, administering miR-146 mimic to males attenuated lung damage, whereas administering miR-146 inhibitor to females increased pulmonary injury.

Background: Ameloblasts are epithelially derived cells responsible for enamel formation through a process known as amelogenesis. Amongst the several transcription factors that are expressed during amelogenesis, both Msx2 and Sp6 transcription factors play important role. Msx2 and Sp6 mouse mutants, exhibit similar amelogenesis defects, namely enamel hypoplasia, while humans with amelogenesis imperfecta AI carry mutations in the human homologues of MSX2 or SP6 genes. These across species similarities in function indicate that these two transcription factors may reside in the same developmental pathway. In this paper, we test whether they work in a coordinated manner to exert their effect during amelogenesis. Methods: Two different dental epithelial cell lines, the mouse LS8 and the rat G5 were used for either overexpression or silencing of Msx2 or Sp6 or both. Msx2 mutant mouse embryos or pups were used for in vivo studies. In situ hybridization, semi-quantitative and quantitative real time PCR were employed to study gene expression pattern. MatInspector was used to identify several potential putative Msx2 binding sites upstream of the murine Sp6 promoter region. Chromatin Immunoprecipitation was used to confirm the binding of Msx2 to Sp6 promoter at the putative sites. Results: Using the above methods we identified that (i) Msx2 and Sp6 exhibit overlapping expression in secretory ameloblasts (ii) Sp6 expression is reduced in the Msx2 mouse mutant secretoty ameloblasts and (iii) that Msx2, like Sp6 inhibits follistatin expression. Specifically, our loss-of function studies by silencing Msx2 and/or Sp6 in mouse dental epithelial (LS8) cells showed significant downregulation of Sp6 but upregulation of Fst expression. Transient transfection of Msx2 overexpression plasmid, up-regulated Sp6 and downregulated Fst expression. Additionally, using MatInspector, we identified several potential putative Msx2 binding sites, 3.5 Kb upstream of the murine Sp6 promoter region. By Chromatin Immunoprecipitation, we confirmed the binding of Msx2 to Sp6 promoter at these sites, thus suggesting that Sp6 is a direct target of Msx2. Conclusions: Collectively, these results show that Sp6 and Msx2 work in a concerted manner to form part of a network of transcription factors that operate during later stages of tooth development controlling ameloblast life cycle and amelogenesis.

MicroRNAs (miRs) have been shown to disrupt normal lung development and function by interrupting alveolarization and vascularisation leading to development of bronchopulmonary dysplasia (BPD). Here we report that miR-184 has a critical role in the induction of BPD phenotype characterised by abnormal alveolarization and pulmonary angiogenesis in the developing lung. We observed an increased expression of miR-184 in BPD clinical specimens: tracheal aspirates (TA), human neonatal lungs with BPD and in fetal human lung Type II alveolar epithelial cells (TIIAECs) exposed to hyperoxia. Consistent with this, we also detected an upregulated miR-184-3p expression in whole lungs, in freshly isolated TIIAECs from lungs of hyperoxia-induced experimental BPD mice and in fetal mice lung TIIAECs exposed to hyperoxia. We demonstrate that overexpression of miR-184-3p exacerbates the BPD pulmonary phenotype, while downregulation of miR-184-3p expression ameliorated the BPD phenotype and also improved respiratory function. We identified miR-184 specific targets: platelet-derived growth factor-beta (Pdgf-β) and friend of Gata 2 (Fog2), also known as zinc finger protein family member (Zfpm2), and show that they are critically involved in pulmonary alveolarization and angiogenesis. Using cell-based luciferase analysis, downregulation of miR-184-3p expression and gene knockdown of miR-184-3p targets Pdgf-β and Fog2 in lung TIIAECs and endothelial cells, we mechanistically show that inhibition of miR-184-3p expression improves pulmonary alveolarization by regulating PDGF-β/AKT/Foxo3/Bax, Bcl2 signalling and enhances angiogenesis by Fog2/VEGF-A/Angiopoietin-1/2 pathway. Collectively, these data suggest that the use of miR-184-3p specific inhibitors may act as novel therapeutic interventions to control the adverse effects of hyperoxia on lung development and function.

Bronchopulmonary dysplasia (BPD) is a common respiratory disease of preterm infants. Lower circulatory/intrapulmonary levels of the adipokine, adiponectin (APN), occur in premature and small-for-gestational-age infants and at saccular/alveolar stages of lung development in the newborn rat. However, the role of low intrapulmonary APN during hyperoxia exposure in developing lungs is unknown.We test the hypothesis that treatment of hyperoxia-exposed newborn mice with recombinant APN protein attenuates the BPD phenotype characterized by inflammation, impaired alveolarization, and dysregulated vascularization. We used developmentally appropriate in vitro and in vivo BPD modeling systems as well as human lung tissue.We observed reduced levels of intrapulmonary APN in experimental BPD mice and human BPD lungs. APN-deficient (APNLow intrapulmonary APN is associated with disruption of lung development during hyperoxia exposure, while recombinant APN protein attenuates the BPD pulmonary phenotype.Intrapulmonary APN levels were significantly decreased in lungs of experimental BPD mice and human BPD lung tissue at various stages of BPD development. Correlative data from human lung samples with decreased APN levels were associated with increased lung adhesion markers (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin). Decreased APN levels were associated with endothelial dysfunction and moderate BPD phenotype in APN-deficient, as compared to WT, experimental BPD mice. WT experimental BPD mice treated with recombinant APN protein had an improved pulmonary structural and functional phenotype. Exogenous APN may be considered as a potential therapeutic agent to prevent BPD.

Infants born extremely preterm are at a high risk of developing bronchopulmonary dysplasia (BPD) which is characterized by large, simplified alveoli, increased inflammation, disrupted and dysregulated vasculogenesis, decreased cell proliferation, and increased cell death in the lungs. Due to lack of specific drug treatments to combat this condition, BPD and its long-term complications have taken a significant toll of healthcare resources. AVR-25, a novel immune modulator experimental compound, was able to partially recover the pulmonary phenotype in the hyperoxia-induced experimental mouse model of BPD. We anticipate that AVR-25 will have therapeutic potential for managing human BPD.

Additional file 2: Figure S2. Effect of hyperoxia on expression of miR-451 in MLE12 cells. RNA was extracted from MLE12 cells grown in room air and exposed to hyperoxia (95% O2) for either 4 or 16 h. miR-451 expression was evaluated using RT-qPCR. N = 3, in each group. MLE12 cells: mouse lung epithelial cells, RA: room air, Hyp – 4H: hyperoxia for 4 h; Hyp-16H: hyperoxia for 16 h; p = 0.13. Data are expressed as mean ± SEM.

Additional file 1: Figure S1. miR-451 expression is upregulated in fetal MLECs following exposure to hyperoxia. RNA was extracted from fetal MLECs grown in room air and exposed to hyperoxia (60% O2) for 16 h. miR-451 expression was evaluated using RT-qPCR. N = 3, in each group. Fetal MLECs: fetal mouse lung endothelial cells, RA: room air, Hyp-16H: hyperoxia with 60% O2 for 16 h; RA: room air; * p

Background Invasive mechanical ventilation (IMV) has become one of the mainstays of therapy in NICUs worldwide, as a result of which premature babies with extremely low birth weight have been able to survive. Although lifesaving, IMV can result in lung inflammation and injury. Surfactant therapy is considered a standard of care in preterm infants with immature lungs. Recently, small molecule inhibitors like siRNAs and miRNAs have been used for therapeutic purposes. Ddit3 (CHOP), Ang2 and miR34a are known to be upregulated in experimental lung injury. We wanted to test whether inhibitors for these molecules (CHOP siRNA, Ang2 siRNA, and miR34a antagomir) if used alone or with a combination with surfactant (Curosurf®) would help in reducing ventilation and hyperoxia-induced injury in an experimental lung injury model. Methods Preterm rabbits born by cesarean section were intratracheally instilled with the three small molecule inhibitors with or without Curosurf® prior to IMV and hyperoxia exposure. Prior to testing the inhibitors in rabbits, these small molecule inhibitors were transfected in mouse lung epithelial cells (MLE12 and AECII) and delivered to neonatal mouse pups intranasally as a proof of concept that surfactant (Curosurf®) could be used as an effective vehicle for administration of such drugs. Survival, pulmonary function tests, histopathology, immunostaining, quantitative PCR and western blotting were done to see the adjuvant effect of surfactant with these three small molecule inhibitors. Results Our data shows that Curosurf® can facilitate transfection of small molecules in MLE12 cells with the same and/or increased efficiency as Lipofectamine. Surfactant given alone or as an adjuvant with small molecule inhibitors increases survival, decreases IMV and hyperoxia-induced inflammation, improves pulmonary function and lung injury scores in preterm rabbit kits. Conclusion Our study shows that Curosurf® can be used successfully as an adjuvant therapy with small molecule inhibitors for CHOP/Ang2/miR34a. In this study, of the three inhibitors used, miR34a inhibitor seemed to be the most promising compound to combat IMV and hyperoxia-induced lung injury in preterm rabbits.

Recent evidence has shown that microRNAs (miRs) are involved in endothelial dysfunction and vascular injury in lung-related diseases. However, the potential role of miR-34a in the regulation of pulmonary endothelial dysfunction, vascular injury, and endothelial cells (ECs) apoptosis in acute lung injury (ALI)/acute lung respiratory distress syndrome is largely unknown. Here, we show that miR-34a-5p was upregulated in whole lungs, isolated ECs from lungs, and ECs stimulated with various insults (LPS and hyperoxia). Overexpression of miR-34a-5p in ECs exacerbated endothelial dysfunction, inflammation, and vascular injury, whereas the suppression of miR-34a-5p expression in ECs and miR-34a-null mutant mice showed protection against LPS- and hyperoxia-induced ALI. Furthermore, we observed that miR-34a-mediated endothelial dysfunction is associated with decreased miR-34a direct-target protein, sirtuin-1, and increased p53 expression in whole lungs and ECs. Mechanistically, we show that miR-34a leads to translocation of p53 and Bax to the mitochondrial compartment with disruption of mitochondrial membrane potential to release cytochrome C into the cytosol, initiating a cascade of mitochondrial-mediated apoptosis in lungs. Collectively, these data show that downregulating miR-34a expression or modulating its target proteins may improve endothelial dysfunction and attenuate ALI.

Delineation of groundwater potential zones was carried out using fuzzy geospatial approach for the sustainable groundwater resource management in Raipur district, Chhattisgarh, India. In the present study, eleven groundwater controlling factors were selected and their respective fuzzy membership (FM) layers were overlaid with fuzzy GAMMA operator to develop the groundwater potential zone (GPZ) map. A total of 62.62% and 37.38% of the total area fell under low to moderate and high GPZ category, respectively. Sensitivity analysis highlighted that lineament density (SD = 0.0057), proximity to surface water body (SD = 0.0046), and aquifer (SD = 0.0036) were the significant contributors towards the groundwater potentiality of the region. Ground truth verification of the proposed model was done with available well yield data of the study area and a linear relation between computed GPZ indices and well yield values was found with a significant positive correlation value of 0.77 (r) that justified the model reliability and authenticity. The proposed model is a robust tool and can be utilized with versatile scale dataset in any parts of the world.

Administration of supplemental oxygen remains a critical clinical intervention for survival of preterm infants with respiratory failure. However, prolonged exposure to hyperoxia can augment pulmonary damage, resulting in developmental lung diseases embodied as hyperoxia-induced acute lung injury and bronchopulmonary dysplasia (BPD). We sought to investigate the role of autophagy in hyperoxia-induced apoptotic cell death in developing lungs. We identified increased autophagy signaling in hyperoxia-exposed mouse lung epithelial-12 cells, freshly isolated fetal type II alveolar epithelial cells, lungs of newborn wild-type mice, and human newborns with respiratory distress syndrome and evolving and established BPD. We found that hyperoxia exposure induces autophagy in a Trp53-dependent manner in mouse lung epithelial-12 cells and in neonatal mouse lungs. Using pharmacological inhibitors and gene silencing techniques, we found that the activation of autophagy, upon hyperoxia exposure, demonstrated a protective role with an antiapoptotic response. Specifically, inhibiting regulatory-associated protein of mechanistic target of rapamycin (RPTOR) in hyperoxia settings, as evidenced by wild-type mice treated with torin2 or mice administered (Rptor) silencing RNA via intranasal delivery or Rptor+/−, limited lung injury by increased autophagy, decreased apoptosis, improved lung architecture, and increased survival. Furthermore, we identified increased protein expression of phospho-beclin1, light chain-3-II and lysosomal-associated membrane protein 1, suggesting altered autophagic flux in the lungs of human neonates with established BPD. Collectively, our study unveils a novel demonstration of enhancing autophagy and antiapoptotic effects, specifically through the inhibition of RPTOR as a potentially useful therapeutic target for the treatment of hyperoxia-induced acute lung injury and BPD in developing lungs.

Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product–induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.

Background: Human papilloma virus (HPV) accounts for the most common cause of all virus-associated human cancers. Here, we describe the first graphic user interface (GUI)-based automated tool ‘HPVDetector', for non-computational biologists, exclusively for detection and annotation of the HPV genome based on next-generation sequencing data sets. Methods: We developed a custom-made reference genome that comprises of human chromosomes along with annotated genome of 143 HPV types as pseudochromosomes. The tool runs on a dual mode as defined by the user: a ‘quick mode' to identify presence of HPV types and an ‘integration mode' to determine genomic location for the site of integration. The input data can be a paired-end whole-exome, whole-genome or whole-transcriptome data set. The HPVDetector is available in public domain for download: http://www.actrec.gov.in/pi-webpages/AmitDutt/HPVdetector/HPVDetector.html. Results: On the basis of our evaluation of 116 whole-exome, 23 whole-transcriptome and 2 whole-genome data, we were able to identify presence of HPV in 20 exomes and 4 transcriptomes of cervical and head and neck cancer tumour samples. Using the inbuilt annotation module of HPVDetector, we found predominant integration of viral gene E7, a known oncogene, at known 17q21, 3q27, 7q35, Xq28 and novel sites of integration in the human genome. Furthermore, co-infection with high-risk HPVs such as 16 and 31 were found to be mutually exclusive compared with low-risk HPV71. Conclusions: HPVDetector is a simple yet precise and robust tool for detecting HPV from tumour samples using variety of next-generation sequencing platforms including whole genome, whole exome and transcriptome. Two different modes (quick detection and integration mode) along with a GUI widen the usability of HPVDetector for biologists and clinicians with minimal computational knowledge.

Macrophages, circulating in the blood or concatenated into different organs and tissues constitute the first barrier against any disease. They are foremost controllers of both innate and acquired immunity, healthy tissue homeostasis, vasculogenesis and congenital metabolism. Two hallmarks of macrophages are diversity and plasticity due to which they acquire a wobbling array of phenotypes. These phenotypes are appropriately synchronized responses to a variety of different stimuli from either the tissue microenvironment or - microbes or their products. Based on the phenotype, macrophages are classified into classically activated/(M1) and alternatively activated/(M2) which are further sub-categorized into M2a, M2b, M2c and M2d based upon gene expression profiles. Macrophage phenotype metamorphosis is the regulating factor in initiation, progression, and termination of numerous inflammatory diseases. Several transcriptional factors and other factors controlling gene expression such as miRNAs contribute to the transformation of macrophages at different points in different diseases. Understanding the mechanisms of macrophage polarization and modulation of their phenotypes to adjust to the micro environmental conditions might provide us a great prospective for designing novel therapeutic strategy. In view of the above, this review summarises the activation of macrophages, the factors intricated in activation along with benefaction of macrophage polarization in response to microbial infections, pulmonary toxicity, lung injury and other inflammatory diseases such as chronic obstructive pulmonary dysplasia (COPD), bronchopulmonary dysplasia (BPD), asthma and sepsis, along with the existing efforts to develop therapies targeting this facet of macrophage biology.

Despite substantial clinical advances over the past decades, inflammatory lung diseases are a major cause of morbidity and mortality and have become one of the major public health issues across the world. The World Health Organization positions lung diseases second in epidemiology, mortality, and cost and predicts that about one-fifth deaths will be attributed to lung diseases by 2020. Currently, there are no therapeutic ways to inhibit or reverse the pathobiology of many destructive lung diseases that results in dysfunctional lung renovation. However, recent studies indicate that lung mesenchymal stem cells (MSCs) are triggered by local factors to differentiate into myofibroblasts that contribute to disease progression. Therefore, it is of critical significance to understand the molecular and cellular basis of endogenous lung MSCs participation in lung injury and repair. Interestingly, while the lung exhibits tremendous regenerative capacity, restoration of pulmonary function does not occur in many adult lung diseases. To address this condition emphasis has been increased on the development of cell-based therapies, but due to diverse cell types and functions lung is considered as a recalcitrant candidate for these strategies. Currently, origins and contributions of stem cells are under intense investigation for cell-based therapy and pulmonary remodelling. Specialized microenvironments for resident multipotent MSCs have been identified in many adult tissues and normal differentiation processes of these may be disrupted by pathologic micro-environmental stimuli during disease, epigenetic changes, or genetic alteration, which program their contribution to pathologic expansion at the expense of functional tissue regeneration. MSCs are most widely investigated and clinically tested type of stem cell because of their regenerative capacity to mesoderm/non-mesoderm-derived tissues and they also display immune-enhancing as well as immunosuppressive properties. MSCs especially from human are widely studied as compared to other cells, different early stage clinical and scientific studies show potential for repair and renewal of lung tissues and offer great ability for the treatment of several devastating and incurable lung diseases. Bone marrow derived MSCs (BM-MSCs) are currently tested in clinical trials as a potential therapy in patients with such inflammatory lung diseases. Here, we will review the biology of MSCs, their interaction with molecular and cellular pathways, and their modulation of immune responses in lung disorders. Additionally, we discuss what stem cell therapy offers in specific acute and chronic lung disorders which includes respiratory distress syndrome (RDS), chronic obstructive pulmonary disease (COPD), asthma, fibrosis, bronchopulmonary dysplasia (BPD), and pulmonary hypertension.

Turbidity removal from surface water was investigated in laboratory columns to identify critical hydrogeological factors during bank filtration such as fine content, soil bed depth, hydraulic gradient, grain size (d50) of aquifer materials as well as detention and operation time. Effect of hydrogeological factors on electrical conductivity was also evaluated. With increase in detention and operation time increase in turbidity removal efficiency and electrical conductivity was observed for both aquifer material as well as modified aquifer materials. The turbidity removal efficiency for aquifer material increased from 70% to 87.6% and 62.4%–79.1% with increase in detention time and operation time from 1 h to 7 h respectively. It was found that removal of turbidity increased from 84.1% to 100% with increase in percentage fine content from 7.5% to 17.5%, increased from 90% to 99% with increase in soil bed depth from 25 cm to 85 cm and increased from 74.1% to 92% with decrease in grain size i.e. D50 from 3 mm to 0.17 mm considering detention time of 7 h. Multiple linear regression analysis was used to demonstrate the vital hydro-geological factors affecting turbidity removal. Out of different hydro-geological factors, detention time and fine content were directly proportional to turbidity removal efficiency while turbidity removal efficiency was inversely proportional to hydraulic gradient and mean grain size. Validation of model was done using field study data, done at two river bank filtration sites, which gave satisfactory results and thus ensuring the precision of model developed.

LIM-Homeodomain genes encode a family of proteins defined by the cysteine-rich protein/protein interacting (Lin-11, Isl-1, and Mec-3) LIM domain and a highly conserved DNA-binding domain. Studies in several organisms have shown that these transcriptional regulators control multiple aspects of embryonic development and are responsible for the pathogenesis of several human diseases. Here we report the expression of Islet-1 (Isl-1) in the gastrointestinal epithelium in developing and adult mice. At embryonic day (E) 9.5–10.5, Isl-1 expression was first detected in the ventral gastric mesenchyme, and expression in the dorsal mesenchyme initiated a few days later. Isl-1 expression was first observed in the gastric epithelium at E13.5 and at E14.5 was restricted to the posterior half of the stomach. In the mature stomach, Isl-1 expression was detected only in subsets of enteroendocrine cells. Furthermore, Isl-1 expression in the intestinal epithelium was first detected at E15.5 and was restricted to subpopulations of enteroendocrine cells in adult mice. These expression analyses suggest that Isl-1 might have an early broad role in stomach and intestinal cells and a secondary role in terminal differentiation and/or maintenance of mature enteroendocrine subtypes in the gastrointestinal epithelium.

Dural metastasis of metastatic breast cancer has become an increasingly diagnosed entity due to advanced radiological imaging. We present an autopsy case of a 51-year-old woman who presented with dizziness, had dural metastasis with subdural hematoma from a primary high-grade invasive ductal breast carcinoma. The pathogenesis of dural metastasis in our case was due to hematogenous dissemination while the subdural hematoma was due to destruction of vessels by tumor cells. The postmenopausal age and the high-grade histology of our case according to published literature signify a poor prognosis and would have meant an ante mortem median survival time of less than one year. Several studies have shown that treatment of intracranial metastatic cancer improves survival. Early recognition and diagnosis of symptoms of dural metastasis will alleviate the neurological complications of dural metastatic breast cancer. Our case report attempts to contribute to the understanding of dural metastasis in breast cancer and emphasize the importance of CNS surveillance in the treatment of a systemic primary cancer.

We previously used a yeast-based enhancer trap to identify a strong, retinoic acid response element (RARE). We have now characterized testis and eye transcripts that are adjacent to this regulatory element. Bioinformatics analysis of expressed sequence tag (EST) clones and RNase protection, reverse transcription-PCR, and northern blot assays indicate that these two RNAs are transcribed from the same locus on opposite template strands. This positions the RARE upstream of the testis transcript and downstream of the eye transcript. Additionally, these two RNAs are embedded within the third intron of the 329 kbp gene that encodes the Zinc Finger and BTB domain containing 7C protein (Zbtb7C). We present the evidence indicating that the testis transcript is expressed primarily in spermatocytes and/or early round spermatids. Furthermore, our analyses of transcript levels in eyes and testes isolated from vitamin A deficient mice or from mice with defects in retinoid storage or signaling indicate that retinoids are required for expression in vivo.

Vitamin A is a very common teratogen causing severe embryonic malformations and changes in pattern formation in amphibians. The effects of vitamin A include the induction of duplication of skeletal structures in the anteroposterior, proximodistal and dorsoventral axes. The purpose of this investigation was to study the histological changes in the regenerating tail of the tadpoles of Polypedates maculatus by treatment with vitamin A. The histological changes brought about by vitamin A included the development of multilayered epidermis and thickened basement membrane, enlargement of the notochord and the nerve cord and the clumping of muscles. In addition, single, paired and supernumerary homeotic limbs were visible from day 20 onwards at ectopic sites. Initially, the limb consisted of only procartilage cells. Muscles and digits appeared from day 48 onwards. In contrast, such observations were not found in the control tails. The significance of such changes is discussed.

A penta-nucleotide microsatellite marker (CCTTT)n, in NOS2A promoter region has been associated with a variety of infectious diseases, including Tuberculosis. Most of these studies, however, are limited in justifying the association to tuberculosis through independent functional assays. The present study on 915 individuals from three geographically distinct populations of India focused on the association and function simultaneously. PCR-Polyacrylamide Gel Electrophoresis and direct sequencing revealed the association of (CCTTT)10 allele with protection against Tuberculosis in all the three populations (i) independently: Odisha [p = 0.0025, pc = 0.025, OR (95%CI) = 0.5 (0.39-0.82)], Chhattisgarh-tribe [p = 0.0001, pc = 0.001, OR (95%CI) = 0.3 (0.19-0.60)] and Sahariya tribe [p = 0.02, pc = 0.2, OR (95%CI) = 0.27 (0.08-0.84)]; and (ii) together: [p 0.0001, pc 0.001, OR (95%CI) = 0.47 (0.35-0.63)]. In-vitro dual luciferase reporter assay revealed that (CCTTT)n microsatellite affected the expression of the reporter gene without enhancing the promoter strength in repeat-size specific manner. Thus the genetic association observed did not correlate exactly with the expression pattern and probably depended on flanking promoter SNPs (rs2779249rs140293907) not in strong LD with the microsatellite, and this effect needs future confirmation.

Objectives To study angiogenesis in metastasis of unknown primary (MUP) to support the authors9 hypothesis that MUPs are of a low angiogenic phenotype and are able to undergo metastasis in spite of an unknown primary. Patients and methods A retrospective analysis was performed on paraffin blocks obtained from 50 cases of MUP and 52 cases of metastasis of known primary (MKP) from 1 January 2000 to December 2003. A prospective analysis was performed on fresh tissues from 22 cases of MUP and 26 cases of MKP. Immunohistochemical staining for VEGF was performed on the paraffin blocks. The fresh frozen tissue was analysed by RT-PCR and Western blotting for VEGF isoforms (VEGF 121 , VEGF 165 , VEGF 189 ) and VEGF protein, respectively. Results Immunohistochemistry showed that MUPs had a higher percentage of lower scores of VEGF expression than MKPs. MKPs had increased scores of VEGF expression. RT-PCR analysis showed that MKPs had increased expression of VEGF 121 and VEGF 165 isoforms as compared with MUPs. MKPs showed a higher VEGF protein expression than MUPs. Conclusion The study shows that metastases of squamous carcinoma from unknown primary have decreased VEGF expression at the immunohistochemical and protein level. They also display decreased expression of the VEGF 121 and VEGF 165 isoforms. Hence, these tumours are of a low angiogenic phenotype. They are able to develop a metastatic phenotype and grow at the metastatic site, since angiogenesis is redundant for lymph-node metastasis.

Glial-derived neurotrophic factor (Gdnf) is required for morphogenesis of the enteric nervous system (ENS) and it has been shown to regulate proliferation, differentiation, and survival of cultured enteric neural crest-derived cells (ENCCs). The goal of this study was to investigate its in vivo role in the colon, the site most commonly affected by intestinal neuropathies such as Hirschsprung's disease. Gdnf activity was modulated in ovo in the distal gut of avian embryos using targeted retrovirus-mediated gene overexpression and retroviral vector-based gene silencing. We find that Gdnf has a pleiotropic effect on colonic ENCCs, promoting proliferation, inducing neuronal differentiation, and acting as a chemoattractant. Down-regulating Gdnf similarly induces premature neuronal differentiation, but also inhibits ENCC proliferation, leading to distal colorectal aganglionosis with severe proximal hypoganglionosis. These results indicate an important role for Gdnf signaling in colonic ENS formation and emphasize the critical balance between proliferation and differentiation in the developing ENS.