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Background: The pressures of medical school can be extremely difficult for personal wellness and contribute to anxiety, burnout and depression. High demands for academic performance and restraints of extracurricular activities all add to distress of a medical student. Methodology: A cross sectional study using convenient sampling was carried out in medical students of CPMC, Lahore. A sample size of 208 was taken. The data was collected using the SF-36 questionnaire. Data was presented as percentages, frequencies, mean and standard deviation. Results: The mean score of physical activity was 61.35+27.02 SD. The average score of restraints due to physical health and hindrance due to emotional health was 38.58+37.49 SD and 34.26+15.37 SD. The mean score of discomfort was 59.57+27.70 SD. The Mean+SD of the score for vitality or tiredness was 46.47+16.69 SD. The average score of emotional health, social relationships and general fitness was 51.10+16.64 SD, 52.82+21.57 SD and 54.04+18.76 SD. Practical implication of the study is to observe the physical and emotional health of students enrolled in MBBS program. Conclusion: The questionnaire SF-36 has been used in the past literature for the assessment of physical and emotional well-being of patients suffering from any particular disease. We observed that the female participants had poor physical and emotional health compared to male participants. Keywords: Mental, physical, medical students, assessment.

Background: Corona virus belongs to the coronaviridea family. Its variant, the severe acute respiratory coronavirus-2 (SARS-COV-2) is a beta coronavirus causing the current Covid-19 outbreak in humans. This virus not only has pathological effects, but it also affects psychologically. Our study aims to find out the mental changes affecting the COVID recovered patients. Methodology: A cross-sectional study was carried at Central Park Medical College from July 2021- August 2021 to assess the psychological stress and anxiety among COVID-19 recovered patients. Data was collected from 96 COVID-19 recovered patients through a self-designed questionnaire using convenient sampling design. Results: The mean age of the participants was 28.07 + 12.45 SD in years. About 44.8% of the participants were female. Approximately 79% participants were graduated or above. About 52.4% of the participants who were infected from COVID-19 mentioned that they lost their appetite during illness. Nearly 71.4% of the participants mentioned that they were more anxious during the illness. Conclusion: Nearly two third participants had sad feelings. A high percentage of respondents had some level of anxiety. Most of the participants reported sudden and panic attacks after COVID. Many respondents said that they were trying to overcome these mental after affects of COVID.

“It's a men's world women are here only to assist, serve and please men. It’s the way the world has always been.” In the world there is only one supreme culture prevailing universally in all the societies is the men-centric culture, which is all powerful and all pervasive. This patriarchal culture and its norms are so deeply engrained in the soul of people that they relegate women into fringe or secondary position. Women's rights talk about equality in each aspects of life but in India where patriarchy has solid roots in society, it appears to be extremely hard to acquire concordance and equality in power structures. Patriarchy has clasped Indian middle class society in its stereotyped shackles and its hold on society is unbending and heavy which adversely affects the lives of women. The torture born by women is the result of brutal dominating tendency of males and Tendulkar in general, spotlights on the patriarchal society in both of his plays. He indicated how a men centric society and women's liberation are interconnected and how male domination transforms into maltreatment of women. All the power in Indian society is in the hands of the males which at last prompts the pathetic state of females. Power, when gets discordant, without a doubt results into persecution and domination whether it is mastery of man over women or the other way around. Indian middle class has the same imbalanced power structure and consequently male domination exists because of male dominated society. The ruling idea of males tosses women into a well of persecution of each sort. Such state of females is exhibited by Tendulkar in ‘Sakharam Binder' and in ‘Silence the Court is in Session' caused by the patriarchal system of culture wherein the women have to struggle hard and pass through severe plight to establish their place in society. As we see in case of these three women Miss Benare, Laxmi and Champa. All these three are discarded women who strive hard for their existence in society and have to pass through so many inextricable difficulties. But the plight goes immutable, and unending.

Introduction There are several factors such as physical inactivity, sedentary lifestyle, and diet that can be responsible for weight gain or obesity. Regular physical activity is important for better physical and emotional well-being. The objective of the study is to observe the prevalence of obesity or over-weight and how lack of physical activity contributes to weight gain and other health issues. Methods This cross-sectional study was conducted in Shalamar Town, Lahore on 646 participants. Data was collected using the WHO STEPS instrument. The inclusion criteria were a minimum age of 30 years and residents of Shalimar Town, Lahore for more than five years. The exclusion criteria were participants with comorbid conditions like HIV, TB, and terminal stage of cancer. Test of association and binary logistic regression analysis was performed to observe a significant association between demographic variables and non-communicable diseases among the participants involved in performing physical exercise. Results About 22.1% of the participants had normal BMI, 5.3% were underweight whereas 34.2% of the participants were overweight and 32.4% obese. Male participants were found to be more physically active compared to females. Hypertension and diabetes were statistically significantly associated with physical activity. BMI and waist-hip ratio were found to be associated with moderate physical exercise. Conclusion Most of the participants were not involved in moderate physical activity. Overall, an alarming 66.6% of the participants were either overweight or obese. In general, males were found to participate more in intense physical activity.

Expansion of renal lymphatic networks, or lymphangiogenesis (LA), is well recognized during development and is now being implicated in kidney diseases. Although LA is associated with multiple pathological conditions, very little is known about its role in acute kidney injury. The purpose of this study was to evaluate the role of LA in a model of cisplatin-induced nephrotoxicity. LA is predominately regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D, ligands that exert their function through their cognate receptor VEGF receptor 3 (VEGFR3). We demonstrated that use of MAZ51, a selective VEGFR3 inhibitor, caused significantly worse structural and functional kidney damage in cisplatin nephrotoxicity. Apoptotic cell death and inflammation were also increased in MAZ51-treated animals compared with vehicle-treated animals following cisplatin administration. Notably, MAZ51 caused significant upregulation of intrarenal phospho-NF-κB, phospho-JNK, and IL-6. Cisplatin nephrotoxicity is associated with vascular congestion due to endothelial dysfunction. Using three-dimensional tissue cytometry, a novel approach to explore lymphatics in the kidney, we detected significant vascular autofluorescence attributed to erythrocytes in cisplatin alone-treated animals. Interestingly, no such congestion was detected in MAZ51-treated animals. We found increased renal vascular damage in MAZ51-treated animals, whereby MAZ51 caused a modest decrease in the endothelial markers endomucin and von Willebrand factor, with a modest increase in VEGFR2. Our findings identify a protective role for de novo LA in cisplatin nephrotoxicity and provide a rationale for the development of therapeutic approaches targeting LA. Our study also suggests off-target effects of MAZ51 on the vasculature in the setting of cisplatin nephrotoxicity. NEW & NOTEWORTHY Little is known about injury-associated LA in the kidney and its role in the pathophysiology of acute kidney injury (AKI). Observed exacerbation of cisplatin-induced AKI after LA inhibition was accompanied by increased medullary damage and cell death in the kidney. LA inhibition also upregulated compensatory expression of LA regulatory proteins, including JNK and NF-κB. These data support the premise that LA is induced during AKI and lymphatic expansion is a protective mechanism in cisplatin nephrotoxicity.

Sepsis is a significant cause of mortality in hospitalized patients. Concomitant development of acute kidney injury (AKI) increases sepsis mortality through unclear mechanisms. While electrolyte disturbances and toxic metabolite buildup during AKI could be important, it is possible that the kidney produces a protective molecule lost during sepsis with AKI. We previously demonstrated that systemic Tamm-Horsfall Protein (THP, uromodulin), a kidney-derived protein with immunomodulatory properties, falls in AKI. Using a mouse sepsis model without severe kidney injury, we show that the kidney increases circulating THP by enhancing basolateral release of THP from medullary thick ascending limb cells. In sepsis patients, changes in circulating THP are positively associated with critical illness. THP is also found de novo in injured lungs. Genetic ablation of THP in mice leads to increased mortality and bacterial burden during sepsis. Consistent with the increased bacterial burden, the presence of THP in vitro and in vivo leads macrophages and monocytes to upregulate a transcriptional program promoting cell migration, phagocytosis and chemotaxis and treatment of macrophages with purified THP increases phagocytosis. Rescue of septic THP-/- mice with exogenous systemic THP improves survival. Together, these findings suggest that through releasing THP, the kidney modulates the immune response in sepsis by enhancing mononuclear phagocyte function and systemic THP has therapeutic potential in sepsis.Significance StatementSepsis is a significant contributor to kidney injury as well as morbidity and mortality worldwide. Specific therapies to improve outcomes in sepsis with kidney injury have largely been limited to symptom management and infectious agent control, in part because it is unclear how kidney injury increases sepsis mortality. This paper describes the identification of Tamm-Horsfall protein, previously known to protect in ischemic models of AKI, as protective in preclinical models of sepsis. It demonstrates how the loss of THP leads to decreased mononuclear phagocyte function and diversity, increased pathogen burden and decreased survival. THP also increases in sepsis without severe kidney injury and concentrates in injured organs. Further study of THP in sepsis could lead to novel sepsis therapeutics.

Soil pollution with potentially toxic elements is a significant environmental problem worldwide. Such soils need reclamation, utilizing environmentally friendly and cost-effective techniques. Therefore, a study was performed with an aim to evaluate the effect of different organic amendments on bioaccumulation of potentially toxic elements in spinach grown on contaminated soils. For the experiment, soils from various mines (manganese mine (MM), chromite mine (CM), quartz mine (QM), and soap stone mine (SSM)) were amended with organic fertilizers including spent mushroom compost (SMC), vermicompost (VC), and leaf compost (LC). Spinach was grown in pots containing 5 kg growing media at 1:1:1 by weight (garden soil:mining soil:organic fertilizer). Results indicated that mining soils and organic amendments significantly affected plant growth and yield parameters and potentially toxic elements’ content in spinach. Organic fertilizers amendment reduced potentially toxic elements’ availability, increased spinach growth, and reduced their related health risk indices (HRI). Among organic fertilizers, VC was more effective in reducing uptake of lead (Pb), manganese (Mn), chromium (Cr), and cadmium (Cd) by 81%, 59%, 74%, and 45%, respectively; improving spinach growth and yield; and reducing HRI.

The ability to effectively monitor key indicators is important for continuous quality improvement in laboratory immunohistochemistry. This article deals specifically with laboratory turnaround time (TAT) as a key delivery indicator and the impact of laboratory workflow on laboratory TATs. While our laboratory has traditionally relied on the manual calculation of slide-TAT (S-TAT) to monitor delivery, we have determined that automated calculation of case-TAT (C-TAT) would be superior as a delivery indicator. AABACUS (Automatable Activity-Based Approach to Complexity Unit Scoring) is an activity-based workload model designed to function primarily as a decision support tool to monitor pathologist staffing levels. We devised a high-level proof-of-principle approach to determine whether it is possible to apply AABACUS as a decision support tool for quality improvement through analysis of alternative laboratory workflows that have potential to impact C-TAT. Our use of AABACUS in this proof-of-principle quality improvement endeavour was two-fold: (1) we leveraged the ability of AABACUS to link data at the slide level to data at the case level, which enabled the automated calculation of C-TAT; and (2) we adapted AABACUS to evaluate the impact of laboratory workflow activities (specifically workflow bifurcation activities) on the calculated C-TATs. We have coined the term 'L-AABACUS' to describe the adaptation of AABACUS to the analysis of laboratory workflow.

Tamm–Horsfall protein (THP), also known as uromodulin, is a kidney-specific protein produced by cells of the thick ascending limb of the loop of Henle. Although predominantly secreted apically into the urine, where it becomes highly polymerized, THP is also released basolaterally, toward the interstitium and circulation, to inhibit tubular inflammatory signaling. Whether, through this latter route, THP can also regulate the function of renal interstitial mononuclear phagocytes (MPCs) remains unclear, however. Here, we show that THP is primarily in a monomeric form in human serum. Compared with wild-type mice, THP(−/−) mice had markedly fewer MPCs in the kidney. A nonpolymerizing, truncated form of THP stimulated the proliferation of human macrophage cells in culture and partially restored the number of kidney MPCs when administered to THP(−/−) mice. Furthermore, resident renal MPCs had impaired phagocytic activity in the absence of THP. After ischemia-reperfusion injury, THP(−/−) mice, compared with wild-type mice, exhibited aggravated injury and an impaired transition of renal macrophages toward an M2 healing phenotype. However, treatment of THP(−/−) mice with truncated THP after ischemia-reperfusion injury mitigated the worsening of AKI. Taken together, our data suggest that interstitial THP positively regulates mononuclear phagocyte number, plasticity, and phagocytic activity. In addition to the effect of THP on the epithelium and granulopoiesis, this new immunomodulatory role could explain the protection conferred by THP during AKI.

Analysis of the immune system in the kidney relies predominantly on flow cytometry. Although powerful, the process of tissue homogenization necessary for flow cytometry analysis introduces bias and results in the loss of morphologic landmarks needed to determine the spatial distribution of immune cells. An ideal approach would support three-dimensional (3D) tissue cytometry: an automated quantitation of immune cells and associated spatial parameters in 3D image volumes collected from intact kidney tissue. However, widespread application of this approach is limited by the lack of accessible software tools for digital analysis of large 3D microscopy data. Here, we describe Volumetric Tissue Exploration and Analysis (VTEA) image analysis software designed for efficient exploration and quantitative analysis of large, complex 3D microscopy datasets. In analyses of images collected from fixed kidney tissue, VTEA replicated the results of flow cytometry while providing detailed analysis of the spatial distribution of immune cells in different regions of the kidney and in relation to specific renal structures. Unbiased exploration with VTEA enabled us to discover a population of tubular epithelial cells that expresses CD11C, a marker typically expressed on dendritic cells. Finally, we show the use of VTEA for large-scale quantitation of immune cells in entire human kidney biopsies. In summary, we show that VTEA is a simple and effective tool that supports unique digital interrogation and analysis of kidney tissue from animal models or biobanked human kidney biopsies. We have made VTEA freely available to interested investigators via electronic download.

High serum concentrations of kidney-derived protein uromodulin (Tamm-Horsfall protein or THP) have recently been shown to be independently associated with low mortality in both older adults and cardiac patients, but the underlying mechanism remains unclear. Here, we show that THP inhibits the generation of reactive oxygen species (ROS) both in the kidney and systemically. Consistent with this experimental data, the concentration of circulating THP in patients with surgery-induced acute kidney injury (AKI) correlated with systemic oxidative damage. THP in the serum dropped after AKI, and was associated with an increase in systemic ROS. The increase in oxidant injury correlated with post-surgical mortality and need for dialysis. Mechanistically, THP inhibited the activation of the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) channel. Furthermore, inhibition of TRPM2 in vivo in a mouse model, mitigated the systemic increase in ROS during AKI and THP deficiency. Our results suggest that THP is a key regulator of systemic oxidative stress by suppressing TRPM2 activity and our findings might help to explain how circulating THP deficiency is linked with poor outcomes and increased mortality.

The present study is an attempt to evaluate environmental awareness and attitude among the slum dwellers. Results based upon dwellers responses to various questions on environmental issues revealed that majority of the respondents (76.19%) had score indicating high level of basic environmental awareness. Among the gender groups, significant difference between the basic environmental awareness level among male and female respondents was observed but no significant difference was shown among different educational and age groups. Similarly, majority of respondents showed a high level (86.81%) of environmental attitude.

Sniffers possess huge potential in monitoring and validating internet traffic. They are essential for efficient working of computer networks in any organization. Sniffer helps to monitor a network as well as capture the passwords used in telnet, login and ftp connections. It examines and sniffs packet travelling through a network without altering them. Sniffer decodes the captured packets and allows network administrators to understand, analyze and recover useful information from packet content. Besides being a boon for organization, hackers also use sniffers for indulging into malicious acts of hacking other users’ account and systems. This paper focuses on the development and implementation of a new sniffer named as Sniflyzer and briefly explores the protocols used in analyzing a packet. Implementation results illustrate that Sniflyzer creates an encrypted database of packet and the front end displays the front end displays the appropriate information as desired by the user.

Background: Tamm-Horsfall Protein (THP) is a glycoprotein expressed exclusively by cells of the thick ascending loop (TAL) of Henle. THP has a protective role in acute kidney injury (AKI), and its expression is downregulated in the early stages of injury. Tumor necrosis factor alpha (TNFE) is a cytokine endogenously expressed by the TAL and is also induced by AKI. Therefore, we hypothesized that TNFE is a key regulator of THP expression. Methods: We used a mouse model of AKI (ischemia-reperfusion injury, IRI) and a cell culture system of a TAL cell line (MKTAL). Results: We show that TNFE is upregulated by TAL cells early after AKI in vivo. The expression of THP and its transcription factor Hepatocyte nuclear factor 1F (HNF1F) were concomitantly decreased at the peak of injury. Furthermore, recombinant TNFE inhibits significantly, and in a dose-dependent manner, the expression of THP, but not HNF1F in MKTAL cells. Interestingly, neither TNFE neutralization nor genetic deletion of TNFE increased THP or HNF levels after injury in vivo. Conclusion: Our data suggest that TNFE can inhibit the expression of THP in TAL cells via an HNF1F-independent mechanism, but the downregulation of THP expression in the early AKI does not depend on TNFE. We propose that TNFE regulates THP expression in a homeostatic setting, but the impact of TNFE on THP during kidney injury is superseded by other factors that could inhibit HNF1F-mediated expression of THP. i 2014 S. Karger AG, Basel

FA is a genetic disorder characterized by BM failure, developmental defects, and cancer predisposition. Previous studies suggest that FA patients exhibit alterations in immunologic function. However, it is unclear whether the defects are immune cell-autonomous or secondary to leukopenia from evolving BM failure. Given the central role that macrophages have in the innate immune response, inflammation resolution, and antigen presentation for acquired immunity, we examined whether macrophages from Fancc−/− mice exhibit impaired function. Peritoneal inflammation induced by LPS or sodium periodate resulted in reduced monocyte/macrophage recruitment in Fancc−/− mice compared with WT controls. Fancc−/− mice also had decreased inflammatory monocytes mobilized into the peripheral blood after LPS treatment compared with controls. Furthermore, Fancc−/− peritoneal macrophages displayed cell-autonomous defects in function, including impaired adhesion to FN or endothelial cells, reduced chemoattractant-mediated migration, and decreased phagocytosis. Moreover, dysregulated F-actin rearrangement was detected in Fancc−/− macrophages after adhesion to FN, which was consistent with an observed reduction in RhoA-GTP levels. Importantly, these data suggest that impaired cytoskeletal rearrangements in Fancc−/− macrophages may be the common mechanism responsible for cell-autonomous defects detected in vitro, as well as altered monocyte/macrophage trafficking in vivo.

Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis . Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of ∼30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.

Tamm-Horsfall protein (THP) is a glycoprotein uniquely expressed in the kidney. We recently showed an important role for THP in mediating tubular cross-talk in the outer medulla and in suppressing neutrophil infiltration after kidney injury. However, it remains unclear whether THP has a broader role in neutrophil homeostasis. In this study, we show that THP deficiency in mice increases the number of neutrophils, not only in the kidney but also in the circulation and in the liver, through enhanced granulopoiesis in the bone marrow. Using multiplex ELISA, we identified IL-17 as a key granulopoietic cytokine specifically upregulated in the kidneys but not in the liver of THP(-/-) mice. Indeed, neutralization of IL-17 in THP(-/-) mice completely reversed the systemic neutrophilia. Furthermore, IL-23 was also elevated in THP(-/-) kidneys. We performed real-time PCR on laser microdissected tubular segments and FACS-sorted renal immune cells and identified the S3 proximal segments, but not renal macrophages, as a major source of increased IL-23 synthesis. In conclusion, we show that THP deficiency stimulates proximal epithelial activation of the IL-23/IL-17 axis and systemic neutrophilia. Our findings provide evidence that the kidney epithelium in the outer medulla can regulate granulopoiesis. When this novel function is added to its known role in erythropoiesis, the kidney emerges as an important regulator of the hematopoietic system.

Although excess hepatic iron in hereditary haemochromatosis and dietary iron overload in the African causes hepatocellular carcinoma, it usually does so in the presence of cirrhosis. A direct hepatocarcinogenic effect of iron has not been proved. Moreover, an animal model of hepatocellular carcinoma induced by iron overload has not been available. The aim of this study was to develop such a model and to use it to ascertain whether excess hepatic iron is directly hepatocarcinogenic. Sixty Wistar albino rats were fed a chow diet and 60 the same diet supplemented initially with 2% carbonyl iron for 12 months, followed by 0.5% ferrocene for 20 months. Five rats from each group were sacrificed every 4 months for 24 months for histological and biochemical monitoring. By 16 months, hepatocytes in all the rats receiving the iron-supplemented diet showed grade 4 iron overload, comparable in degree with that seen in patients with advanced hereditary haemochromatosis and dietary iron overload. Altered hepatic foci and pre-neoplastic nodules were first seen at 16 months. These increased in size and number with time, were iron-free, stained positively with placental glutathione sulphydryl transferase, and showed the same histological features as the iron-free foci described in patients with hepatocellular carcinoma complicating hereditary haemochromatosis. At 32 months the eight surviving rats in the iron overloaded group were sacrificed. The livers of five of these rats contained pre-neoplastic nodules and one showed, in addition, an iron-free, well-differentiated hepatocellular carcinoma. The tumour stained positively for placental glutathione sulphydryl transferase. Neither cirrhosis nor portal fibrosis was present in this or any iron-loaded animal. We conclude that hepatocellular carcinoma may complicate dietary hepatic iron overload in Wistar albino rats in the absence of fibrosis or cirrhosis, confirming an aetiological association between dietary iron overload and the tumour and suggesting that iron may be directly hepatocarcinogenic.

Background Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray. Results Significantly (p < 0.01) improved DNA retention was achieved printing in 15% DMSO/1.5 M betaine compared to the vendor recommended buffers. Introduction of tracking oligonucleotide did not effect hybridization efficiency or introduce ratio measurement bias in hybridizations between M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p < 0.05) and there were no significant differences in Pearson's correlation coefficients of ratio data between replicates possessing (0.72 ± 0.07), replicates lacking (0.74 ± 0.10), or replicates with and without (0.70 ± 0.04) the tracking oligonucleotide. ANOVA analysis confirmed the tracking oligonucleotide introduced no bias. Titrating target-specific oligonucleotide (40 μM to 0.78 μM) in the presence of 0.5 μM tracking oligonucleotide, revealed a fluorescein fluorescence inversely related to target-specific oligonucleotide molarity, making tracking oligonucleotide signal useful for quality control measurements and differentiating false negatives (synthesis failures and mechanical misses) from true negatives (no gene expression). Conclusions This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.

Laser micro-dissection (LMD) is a very useful tool that allows the isolation of finite areas from tissue specimens for downstream analysis of RNA and protein. Although LMD has been adapted for use in kidney tissue, the use of this powerful tool has been limited by the diminished ability to identify specific tubular segments in the kidney. In this study, we describe a major improvement in the methodology to isolate specific cells in the mouse kidney using immunofluorescence LMD (IF-LMD). Using IF-LMD, we can reproducibly isolate not only glomeruli, but also S1–S2 proximal segments, S3 tubules, and thick ascending limbs. We also demonstrate the utility of a novel rapid immunofluorescence staining technique, and provide downstream applications for IF-LMD such as real-time PCR and cutting-edge proteomic studies. This technical breakthrough may become an invaluable tool for understanding cellular and molecular events in the heterogeneous kidney milieu.

The genome of the flowering plant Arabidopsis thaliana has five chromosomes. Here we report the sequence of the largest, chromosome 1, in two contigs of around 14.2 and 14.6 megabases. The contigs extend from the telomeres to the centromeric borders, regions rich in transposons, retrotransposons and repetitive elements such as the 180-base-pair repeat. The chromosome represents 25% of the genome and contains about 6,850 open reading frames, 236 transfer RNAs (tRNAs) and 12 small nuclear RNAs. There are two clusters of tRNA genes at different places on the chromosome. One consists of 27 tRNA(Pro) genes and the other contains 27 tandem repeats of tRNA(Tyr)-tRNA(Tyr)-tRNA(Ser) genes. Chromosome 1 contains about 300 gene families with clustered duplications. There are also many repeat elements, representing 8% of the sequence.

Construction methodologies for cDNA microarrays lack the ability to determine array integrity prior to hybridization, leaving the array itself a source of uncontrolled experimental variation. We solved this problem through development of a three-color cDNA array platform whereby printed probes are tagged with fluorescein and are compatible with Cy3 and Cy5 target labeling dyes when using confocal laser scanners possessing narrow bandwidths. Here we use this approach to: (i) develop a tracking system to monitor the printing of probe plates at predicted coordinates; (ii) define the quantity of immobilized probe necessary for quality hybridized array data to establish pre-hybridization array selection criteria; (iii) investigate factors that influence probe availability for hybridization; and (iv) explore the feasibility of hybridized data filtering using element fluorescein intensity. A direct and significant relationship (R2 = 0.73, P < 0.001) between pre-hybridization average fluorescein intensity and subsequent hybridized replicate consistency was observed, illustrating that data quality can be improved by selecting arrays that meet defined pre-hybridization criteria. Furthermore, we demonstrate that our three-color approach provides a means to filter spots possessing insufficient bound probe from hybridized data sets to further improve data quality. Collectively, this strategy will improve microarray data and increase its utility as a sensitive screening tool.