Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and ≤1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm³ pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 μg/ml), fibronectin (10 μg/ml), and BSA (10 μg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37°C in 5% CO₂ humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.Download video file.^{(144M, mp4)}     

A Study on the Transfer of Iron in Soil–Plant–Animal Continuum Under Semi-arid Environmental Conditions in Sargodha,Pakistan

The present investigation on the iron (Fe) transfer from soil to plant and in turn to animal (cows), as a function of sampling periods was conducted at the Livestock Experimental Station Sargodha, Pakistan which falls under semi-arid conditions. Although the iron transfer from soil to forage increased consistently, the forage Fe content decreased progressively with increase in sampling period. Highest Fe transfer from forage to cow blood plasma was observed during October and lowest during January. The transfer of Fe from forage to animal milk was maximum during the months of October and January and minimum during December. The transfer of Fe to plasma and milk was found to be dependent variably on the growth stage of forage in this investigation. Based on the findings of the present study, it is evident that mineral supplementation with higher Fe availability is urgently warranted to the animals particularly during the months of December and January to enhance plasma Fe in the cows being reared at that livestock farm during the entire grazing period. Thus, obligatory supplementation of Fe to the ruminants is highly recommended. Since the processes involved in iron management system in humans, animals, and plants are basically similar, appropriate elemental management must be provided to the living organisms, otherwise deficient or excessive levels of iron may deteriorate the developing cells of the organisms.     

Salicylic Acid Modulates Antioxidant System,Defense Metabolites,and Expression of Salt Transporter Genes in Pisum sativum Under Salinity Stress

At present plants continuously exposed to salinity stress due to the challenging environment that has reduced the crop growth and productivity worldwide. Application of phytohormones by using seed priming method emerges as one of the most reliable and cost effective to alleviate the toxic effect of salinity stress. In this study, we evaluate the effect of seed-primed salicylic acid (SA) to reduce the adverse effect of different salt concentrations (0, 100, 200, and 300 mM NaCl) in pea (Pisum sativum L.) seedlings. After seedling emergence, percent seed germination was calculated; however, after 60 days; plants were sampled for studying the growth and photosynthetic traits, lipid peroxidation level, antioxidant activities, ions accumulation, and its sequestration. The results depicted that salinity treatments hampered overall growth performance and induced oxidative stress in a dose-dependent manner. Salinity also has negatively influence on ion accumulation as Na⁺ ion increased while K⁺ ion decreased. On the other hand, seed priming with SA significantly reduced the salinity-induced effects on the overall performance of plants, including growth and photosynthetic attributes. SA alleviated the adverse effect of salinity even at higher salinity level by inducing enzymatic and non-enzymatic antioxidant systems, soluble sugars, and proline accumulation, and regulating ion homeostasis along with up-regulation of Na⁺/H⁺ antiporters (SOS1 and NHX1). Thus, seed priming with SA shows a comprehensive role in mitigation of salinity stress and can be used as a model for promising salinity tolerant cultivation.

     

SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD⁺-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein β (C/EBPβ) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.     

Role of ACE and PAI-1 Polymorphisms in the Development and Progression of Diabetic Retinopathy

In the present study we determined the association of angiotensin converting enzyme (ACE) and plasminogen activator inhibitor-1 (PAI-1) gene polymorphisms with diabetic retinopathy (DR) and its sub-clinical classes in Pakistani type 2 diabetic patients. A total of 353 diabetic subjects including 160 DR and 193 diabetic non retinopathy (DNR) as well as 198 healthy controls were genotyped by allele specific polymerase chain reaction (PCR) for ACE Insertion/Deletion (ID) polymorphism, rs4646994 in intron 16 and PAI-1 4G/5G (deletion/insertion) polymorphism, rs1799768 in promoter region of the gene. To statistically assess the genotype-phenotype association, multivariate logistic regression analysis was applied to the genotype data of DR, DNR and control individuals as well as the subtypes of DR. The ACE genotype ID was found to be significantly associated with DR (p = 0.009, odds ratio (OR) 1.870 [95% confidence interval (CI) = 1.04–3.36]) and its sub-clinical class non-proliferative DR (NPDR) (p = 0.006, OR 2.250 [95% CI = 1.098–4.620]), while PAI polymorphism did not show any association with DR in the current cohort. In conclusion in Pakistani population the ACE ID polymorphism was observed to be significantly associated with DR and NPDR, but not with the severe form of the disease i.e. proliferative DR (PDR).     

Effectiveness of potassium sulfate in mitigating salt-induced adverse effects on different physio-biochemical attributes in sunflower (Helianthus annuus L.)

To assess whether foliar application of K+S as potassium sulfate (K₂SO₄) could alleviate the adverse effects of salt on sunflower (Helianthus annuus L. cv. SF-187) plants, a greenhouse experiment was conducted. There were two NaCl levels (0 and 150 mM) applied to the growth medium and six levels of K+S as K₂SO₄ (NS (no spray), WS (spray of water+0.1% Tween 20 solution), 0.5% K+0.21% S, 1.0% K+0.41% S, 1.5% K+0.62% S, and 2.0% K+0.82% S in 0.1% Tween-20 solution) applied two times foliarly to non-stressed and salt-stressed sunflower plants. Salt stress markedly repressed the growth, yield, photosynthetic pigments, water relations and photosynthetic attributes, quantum yield (F_v/F_m), leaf and root K⁺, Mg²⁺, P, Ca²⁺, N as well as K⁺/Na⁺ ratios, while it enhanced the cell membrane permeability, and leaf and root Na⁺ and Cl⁻ concentrations. Foliar application of potassium sulfate significantly improved growth, achene yield, photosynthetic and transpiration rates, stomatal conductance, water use efficiency, leaf turgor and enhanced shoot and leaf K⁺ of the salt-stressed sunflower plants, but it did not improve leaf and root Na⁺, Cl⁻, Mg²⁺, P, Ca²⁺, N as well as K⁺/Na⁺ ratios. The most effective dose of K+S for improving growth and achene yield was found to be 1.5% K+0.62% S and 1% K+0.41% S, respectively. Improvement in growth of sunflower plants due to exogenously applied K₂SO₄ was found to be linked to enhanced photosynthetic capacity, water use efficiency, leaf turgor and relative water content.     

Genetic instability in EBV-transformed lymphoblastoid cell lines

Epstein Barr virus (EBV)-transformed lymphoblastoid cell lines are commonly used to provide an inexhaustible supply of DNA. We examined microsatellite instability in these cell lines in 35 individuals where DNA was available from the original blood samples and from cultured cell lines. Mutations were observed in 0.3% of the analyses, thus providing a quantitative measure of somatic mutation rate.