Use of scanning electron microscopy and energy dispersive X-ray for urine analysis: A preliminary investigation.

Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) are powerful tools to study the ultrastructure of numerous specimens and to determine their elemental composition, respectively. However, results have not yet been reported on their application to urine samples in routine clinical laboratory practice. Herein we investigate urine sediment by using SEM and EDX to detect and identify different urine components. A total of 206 urine samples from patients with and without urinary tract infections were analyzed using SEM and EDX. Microorganisms, crystals, epithelial cells, leukocytes, and erythrocytes were targeted in urine sediment samples. The identification of urine components was based on their morphology, size, contrast, and elemental composition. SEM-analysis allowed us to identify and classify microorganisms in urine sediments into the categories of gram-negative bacilli, cluster cocci, chain cocci, gram-negative bacilli, gram-positive bacilli, and yeasts. In addition, various types of epithelial cells such as renal, transitional, and squamous epithelial cells were found. Furthermore, leukocytes and erythrocytes were well identified, with the detection of various morphological forms of erythrocytes, such as dysmorphic and isomorphic erythrocytes. Using SEM-EDX analysis, calcium oxalate was the most frequently-identified crystal (92.0%), with prominent peaks of C, O, and Ca elements, followed by struvite (6%), with peaks of Mg, P, O, and N. These preliminary data suggest that the two complementary SEM-EDX analyses can be used to detect and identify microorganisms and crystals in urine samples. Further studies are still needed to apply SEM-EDX to urine sediment analysis. SEM-EDX analyses provided comparative results with the routine results, with accurate identification, high resolution and deep focus compared to the routine urinalysis SEM-analysis allowed us to identify and classify microorganisms in urine sediments into the categories of gram-negative bacilli, cluster cocci, chain cocci, gram-negative bacilli, gram-positive bacilli and yeasts. SEM-EDX analysis enabled the accurate identification of crystals based on both morphology and elemental composition.

High-throughput cell death assays.

High-throughput screens (HTS ) are powerful tools that permit the rapid evaluation of thousands of samples in a cost-effective manner and minimize sample and reagent consumption. Such assays have recently begun to be utilized to evaluate cell death modalities and also the cytoprotective efficacy of compounds against a wide variety of stresses. Here we describe the design, preparation, and undertaking of HTS-appropriate assays that utilize simple and cost-effective fluorophore - and luminescence -based functional readouts of cell viability. These assays permit the examination of 96­384 compounds in a single multiwell plate with highly robust statistical significance at a fraction of the financial and work cost of traditional approaches.

Chronic intermittent but not constant hypoxia decreases NAA/Cr ratios in neonatal mouse hippocampus and thalamus.

Chronic constant hypoxia (CCH) and chronic intermittent hypoxia (CIH) are known to have deleterious effects on the central nervous system. Because of the difference in the pattern of hypoxic exposure, it is possible that the pathological outcome would vary. The N-acetyl aspartate/creatine (NAA/Cr) ratio is a reliable marker of neuronal integrity, and this can be noninvasively measured by proton nuclear magnetic resonance spectroscopy. P2 CD1 mouse pups with their dams were exposed to either CCH, where the Fi(O(2)) was maintained at 11% continuously or to CIH, where the Fi(O(2)) was varied between 21 and 11% every 4 min. P30 mice exposed to intermittent hypoxia for 4 wk demonstrated a significant decrease in the NAA/Cr ratio in the hippocampus and thalamus, which was reversed by a subsequent exposure to 4 wk of normoxia. Meanwhile, mice exposed to 4 wk of constant hypoxia did not demonstrate any differences in their NAA/Cr ratios from controls in these brain regions. These results indicate that an intermittent pattern of hypoxic exposure may have a more adverse effect on neuronal function and integrity than a continuous one. The reversal of NAA/Cr levels to baseline during the return to normoxia indicates that therapeutic strategies targeted at alleviating the intermittent hypoxic stress in diseases, such as obstructive sleep apnea, have the potential for inducing significant neurocognitive recovery in these patients.

Gene expression and phenotypic characterization of mouse heart after chronic constant or intermittent hypoxia.

Chronic constant hypoxia (CCH), such as in pulmonary diseases or high altitude, and chronic intermittent hypoxia (CIH), such as in sleep apnea, can lead to major changes in the heart. Molecular mechanisms underlying these cardiac alterations are not well understood. We hypothesized that changes in gene expression could help to delineate such mechanisms. The current study used a neonatal mouse model in CCH or CIH combined with cDNA microarrays to determine changes in gene expression in the CCH or CIH mouse heart. Both CCH and CIH induced substantial alterations in gene expression. In addition, a robust right ventricular hypertrophy and cardiac enlargement was found in CCH- but not in CIH-treated mouse heart. On one hand, upregulation in RNA and protein levels of eukaryotic translation initiation factor-2alpha and -4E (eIF-2alpha and eIF-4E) was found in CCH, whereas eIF-4E was downregulated in 1- and 2-wk CIH, suggesting that eIF-4E is likely to play an important role in the cardiac hypertrophy observed in CCH-treated mice. On the other hand, the specific downregulation of heart development-related genes (e.g., notch gene homolog-1, MAD homolog-4) and the upregulation of proteolysis genes (e.g., calpain-5) in the CIH heart can explain the lack of hypertrophy in CIH. Interestingly, apoptosis was enhanced in CCH but not CIH, and this was correlated with an upregulation of proapoptotic genes and downregulation of anti-apoptotic genes in CCH. In summary, our results indicate that 1) the pattern of gene response to CCH is different from that of CIH in mouse heart, and 2) the identified expression differences in certain gene groups are helpful in dissecting mechanisms responsible for phenotypes observed.