Methods for quantitative and qualitative evaluation of vaginal microflora during menstruation

The quantitative and qualitative changes in the bacterial flora of the vagina during menstruation have received inadequate study. Similarly, the effect of vaginal tampons on the microbial flora as well as the relationship between the microbial flora of the vagina and that of the tampon has not been adequately evaluated. The purposes of the present study were (i) to develop quantitative methods for studying the vaginal flora and the flora of tampons obtained during menstruation and (ii) to determine whether there were differences between the microflora of the tampon and that of the vaginal vault. Tampon and swab samples were obtained at various times from eight young healthy volunteers for 8 to 10 menstrual cycles. Samples consisted of swabs from women wearing menstrual pads compared with swab and tampon samples taken at various times during the menstrual cycle. Samples were analyzed for total facultative and anaerobic bacterial counts, and the six dominant bacterial species in each culture were identified. Statistical evaluation of the results indicates that total bacterial counts decreased during menstruation and that swab and tampon samples yielded similar total counts per unit weight of sample. The numbers of bacteria in tampons tended to be lower than in swabs taken at the same time. Overall, during menstruation, the concentrations of lactobacilli declined, but otherwise there was little difference among the species found during menstruation compared with those found in intermenstrual samples. Cotton tampons had little discernible effect on the microbial flora.     

Methods and conclusions in functional analysis: a reply

This paper is a reply to criticism presented by C. R. C. Paul and R. Cowen in immediately preceding articles in Lethaia. Contrary to the assertion by Paul, the present author did not reject the paradigmatic method but (1) criticized it for its limitations and seemingly erroneous results, (2) applied it to the richthofeniacean and lyttoniacean brachiopods by use of the pump as paradigm, and (3) extended it to include anatomic, metabolic, and phylogenetic considerations that go beyond the structural and mechanistic constraints of the original formulation. The rhythmic flow mechanism was rejected primarily on the basis of evidence for a ptycholophous lophophore in the Productidina. and the relative inefficiency of 'valve flapping' in contrast to ciliary action in pumping nutrient-bearing fluid into the shell. A Chinese blast furnace was claimed by Cowen to employ a single oscillating panel as a pump, but instead this panel must act as a valve that admits air in surges while the draft in the furnace maintains unidirectional flow through the system. This is analogous to ciliary feeding in brachiopods, where undirectional flow is maintained by ciliary pumping, and the valve opens to admit water. A richthofeniid with a Composita entrapped beneath the apertural meshwork is introduced as additional evidence against the likelihood of 'valve flapping'.     

Radiolabeled constructs for evaluation of the asialoglycoprotein receptor status and hepatic functional reserves

Transplantation of isolated hepatocytes may eventually replace a whole liver transplantation for the treatment of selected liver metabolic disorders and acute hepatic failure. To understand the behavior of transplanted hepatocytes, methods for longitudinal assessment of functional activity and survival of hepatocyte transplants must be developed. Targeting of asialoglycoprotein receptor (ASGPr) with various radiolabeled or Gd-labeled constructs of asialofetuin (AF) is expected to allow noninvasive and quantitative assessments of the ASGPr status in functioning hepatocytes before and after the transplant. Six new constructs of (125)I-, (99m)Tc-, (153)Gd-, and (111)In-radiolabeled AF with distinct stabilities and clearance rates were prepared and evaluated in vitro in mice, rat, porcine, and human hepatocytes, and in vivo in mice and rats. The blood and organ clearance rates, as well as liver and spleen uptake, were measured. Even extensive chemical modifications of AF with poly-l-lysine and various chelating agents do not appear to diminish AF's binding to ASGPr. Binding to isolated hepatocytes and the in vivo liver uptake studies indicate unimpaired functional activity of AF as evidenced by the rapid (<10 min) and nearly complete hepatic extraction of AF constructs from the systemic circulation. The catabolic processing and elimination of AF constructs from liver depend on the chemical modification used in the preparation of a given reagent. Radioiodinated AF has by far the shortest postabsorption (5.1 min +/- 0.05 min) and elimination half-lives (2.8 +/- 0.06 h) in liver. In comparison, the AF construct prepared by conjugation of DTPA- and 2-iminothiolane-substituted p-Lys with N-sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (SMPB)-modified AF (AF-SMPB-Traut-p-Lys-((111)In-DTPA)(20)(-)(30)) has a hepatic postabsorption time of 9.1 +/- 0.1 min and an elimination half-life of 44.3 +/- 3.08 h, whereas [(99m)Tc]technetium-labeled AF appears to be permanently retained in liver. These differences in rates of liver uptake and clearance of catabolized radiolabeled AF can be used to determine functional activity of liver and transplanted hepatocytes.     

Methods for quantitative and qualitative evaluation of vaginal microflora during menstruation.

The quantitative and qualitative changes in the bacterial flora of the vagina during menstruation have received inadequate study. Similarly, the effect of vaginal tampons on the microbial flora as well as the relationship between the microbial flora of the vagina and that of the tampon has not been adequately evaluated. The purposes of the present study were (i) to develop quantitative methods for studying the vaginal flora and the flora of tampons obtained during menstruation and (ii) to determine whether there were differences between the microflora of the tampon and that of the vaginal vault. Tampon and swab samples were obtained at various times from eight young healthy volunteers for 8 to 10 menstrual cycles. Samples consisted of swabs from women wearing menstrual pads compared with swab and tampon samples taken at various times during the menstrual cycle. Samples were analyzed for total facultative and anaerobic bacterial counts, and the six dominant bacterial species in each culture were identified. Statistical evaluation of the results indicates that total bacterial counts decreased during menstruation and that swab and tampon samples yielded similar total counts per unit weight of sample. The numbers of bacteria in tampons tended to be lower than in swabs taken at the same time. Overall, during menstruation, the concentrations of lactobacilli declined, but otherwise there was little difference among the species found during menstruation compared with those found in intermenstrual samples. Cotton tampons had little discernible effect on the microbial flora.     

A simple method for the quantitative evaluation of functional effects of xenobiotics with cultured cells

We present a simple, noninvasive, nondestructive all-purpose method for the quantitative evaluation of functional effects of xenobiotics with cultured cells and the work station for its routine, easy implementation. At present 1 to 150 cells growing in one to six dishes can be studied in parallel or otherwise at time intervals ranging from 10 s to 6 h or more, over periods of time ranging from a few tens of minutes to 3–4 days. Any aspect of cell physiological behavior can be studied (differentiation-dedifferentiation, migration, division, degeneration, death) without preliminary staining and/or fixation provided it results in optically visible changes.     

Use of regression analysis in the quantitative evaluation of the activity of biological preparations]

Use of regression analysis in the assessment of the activity of biological preparations under experimental conditions permitted not only to assess the quantitative effect (ED50) more strictly, but also to find other parameters of importance for the results of comparison, for example with the standard, i.e. in standardization. To these belong regression coefficient, parallelism of regressions, and the relative potency. By the presence of a parallelism one can judge the similarity between the activity mechanism of the active principle of the preparations being compared. Relative potency characterizes the activity of the preparation in the relative values in comparison with the standard, with a statistical evaluation of this value with the aid of the confidence interval. The authors suggest a program for Mir-2 computer facilitating the calculations in using the analystical method which is more objective than the graphic method of assessment of the linear dosage-response curve.     

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO‐DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO‐DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA‐based CHO‐DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self‐organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up‐regulating NCK1 and down‐regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial‐ and endoplasmic reticulum‐mediated cell death pathways by up‐regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1026–1038, 2015     

Methods of chromatography and quantitative analysis of galacto- and phospholipids of plant leaves

Data in the literature on chromatography of glycerolipids of plant leaves on columns of DEAE-cellulose, Sephadex LH-20, silica gel, florisil, in a thin layer of silica gel, paper are generalized. Methods of obtaining chromatographically pure glycerolipids, their subfractionation, and the quantitative analysis of glycerolipids are described. Experimental data are presented on fractionation of lipids of potato leaves on columns of DEAE-cellulose, silica gel, on paper, and the quantitative determination of phospholipids. A method of rapid division of lipids into classes by means of their elution with silica gel KSC by different solvents is described.     

Potential use of spectral image analysis for the quantitative evaluation of estrogen receptors in breast cancer

Evaluation of estrogen receptor (ER) content is an important factor in the choice of therapy and prognosis of breast cancer patients. In this study, we demonstrate a new spectral image analysis technique for objective and quantitative evaluations of stained specimens. The SpectraCube system was used to analyze nuclear antigens in thirteen cases of breast cancer stained by the immunoperoxidase method with hematoxylin counterstain. Spectral imaging segregated the spectrum of diaminobenzidine (DAB) from the background color of hematoxylin and a spectral index was calculated. The spectral index essentially agreed with the pathologist's index (on a scale of 0 to 3) in seven out of the thirteen cases. A substantial number of ER positive pixels was detected in the two cases scored as 0 by the pathologist's index. In a test case scored as 1 by the pathologist's index we detected a significant number of pixels, representing 47% of the nuclei, with DAB-intensity values higher than the cut-off value of 1.2. These data suggest that spectral image analysis is a sensitive method providing intensive information with high reproducibility. Our spectral imaging method is highly flexible, enabling the user to define the spatial resolution of the analyzed specimen by choosing the number of pixels per one nucleus.