Biostatistics Series Module 3: Comparing Groups: Numerical Variables

Numerical data that are normally distributed can be analyzed with parametric tests, that is, tests which are based on the parameters that define a normal distribution curve. If the distribution is uncertain, the data can be plotted as a normal probability plot and visually inspected, or tested for normality using one of a number of goodness of fit tests, such as the Kolmogorov–Smirnov test. The widely used Student''s t-test has three variants. The one-sample t-test is used to assess if a sample mean (as an estimate of the population mean) differs significantly from a given population mean. The means of two independent samples may be compared for a statistically significant difference by the unpaired or independent samples t-test. If the data sets are related in some way, their means may be compared by the paired or dependent samples t-test. The t-test should not be used to compare the means of more than two groups. Although it is possible to compare groups in pairs, when there are more than two groups, this will increase the probability of a Type I error. The one-way analysis of variance (ANOVA) is employed to compare the means of three or more independent data sets that are normally distributed. Multiple measurements from the same set of subjects cannot be treated as separate, unrelated data sets. Comparison of means in such a situation requires repeated measures ANOVA. It is to be noted that while a multiple group comparison test such as ANOVA can point to a significant difference, it does not identify exactly between which two groups the difference lies. To do this, multiple group comparison needs to be followed up by an appropriate post hoc test. An example is the Tukey''s honestly significant difference test following ANOVA. If the assumptions for parametric tests are not met, there are nonparametric alternatives for comparing data sets. These include Mann–Whitney U-test as the nonparametric counterpart of the unpaired Student''s t-test, Wilcoxon signed-rank test as the counterpart of the paired Student''s t-test, Kruskal–Wallis test as the nonparametric equivalent of ANOVA and the Friedman''s test as the counterpart of repeated measures ANOVA.     

A randomized cross-over trial to define neurophysiological correlates of AV-101 N-methyl-d-aspartate receptor blockade in healthy veterans

The kynurenine pathway (KP) is a strategic metabolic system that combines regulation of neuronal excitability via glutamate receptor function and neuroinflammation via other KP metabolites. This pathway has great promise in treatment of depression and suicidality. The KP modulator AV-101 (4-chlorokynurenine, 4-Cl-KYN), an oral prodrug of 7-chlorokynurenic acid (7-Cl-KYNA), an N-methyl-d-aspartate receptor (NMDAR) glycine site antagonist, and of 4-chloro-3-hydroxyanthranilic acid (4-Cl-3-HAA), a suppressor of NMDAR agonist quinolinic acid (QUIN), is a promising potential antidepressant that targets glutamate functioning via the KP. However, a recent placebo-controlled clinical trial of AV-101 in depression found negative results. This raises the question of whether AV-101 can penetrate the brain and engage the NMDAR and KP effectively. To address this problem, ten healthy US military veterans (mean age = 32.6 years ± 6.11; 1 female) completed a phase-1 randomized, double-blind, placebo-controlled, crossover study to examine dose-related effects of AV-101 (720 and 1440 mg) on NMDAR engagement measured by γ-frequency band auditory steady-state response (40 Hz ASSR) and resting EEG. Linear mixed models revealed that 1440 mg AV-101, but not 720 mg, increased 40 Hz ASSR and 40 Hz ASSR γ-inter-trial phase coherence relative to placebo. AV-101 also increased 4-Cl-KYN, 7-Cl-KYNA, 4-Cl-3-HAA, 3-HAA, and KYNA in a dose-dependent manner, without affecting KYN and QUIN. AV-101 was safe and well tolerated. These results corroborate brain target engagement of 1440 mg AV-101 in humans, consistent with blockade of interneuronal NMDAR blockade. Future studies should test higher doses of AV-101 in depression. Suicidal behavior, which has been associated with high QUIN and low KYNA, is also a potential target for AV-101.Subject terms: Biomarkers, Neurophysiology, Neuroscience     

Development and Evaluation of Probioticated Cucumber Juice Using <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

The aim of the present study was to develop a probioticated cucumber juice by optimizing the concentration of prebiotic (inulin), stevia (Stevia rebudiana) and inoculum (Lactobacillus plantarum) by varying one factor at a time. Cucumber juice with 2% prebiotic had a pH 3.76, acidity 0.239%, total sugars 198.2 μg/mL, reducing sugars 102.3 μg/mL and microbial viability of 1.36 × 10⁸ colony forming units (CFU)/mL, respectively. The juice with 3% stevia had a pH 3.59, acidity 0.388%, total sugars 214 μg/mL, reducing sugars 156.7 μg/mL and microbial viability of 6.7 × 10⁷ CFU/mL, respectively. The juice with 3% inoculum size had a pH 3.82, acidity 0.39%, total sugars 128.3 μg/mL, reducing sugars 198.6 μg/mL and microbial viability of 3.5 × 10⁸ CFU/mL, respectively. Maximum growth of the probiotic (L. plantarum) in the cucumber juice was obtained at the end of 48 h of fermentation for 2% prebiotic, 3% stevia and 3% inoculum size without much changes in nutritional and organoleptic properties. The probiotic strain namely L. plantarum proved its ability and suitability to ferment cucumber juice. The growth of the probiotic in the cucumber juice was found maximum with 2% prebiotic, 3% stevia and 3% inoculum size at the end of 48 h of fermentation with minimal changes in nutritional value and organoleptic characteristics with a good shelf life at 4 °C in polyethylene terephthalate bottles.     

Sulfated Progesterone Metabolites That Enhance Insulin Secretion via TRPM3 Are Reduced in Serum From Women With Gestational Diabetes Mellitus

Serum progesterone sulfates were evaluated in the etiology of gestational diabetes mellitus (GDM). Serum progesterone sulfates were measured using ultra-performance liquid chromatography–tandem mass spectrometry in four patient cohorts: 1) the Hyperglycemia and Adverse Pregnancy Outcomes study; 2) London-based women of mixed ancestry and 3) U.K.-based women of European ancestry with or without GDM; and 4) 11–13 weeks pregnant women with BMI ≤25 or BMI ≥35 kg/m² with subsequent uncomplicated pregnancies or GDM. Glucose-stimulated insulin secretion (GSIS) was evaluated in response to progesterone sulfates in mouse islets and human islets. Calcium fluorescence was measured in HEK293 cells expressing transient receptor potential cation channel subfamily M member 3 (TRPM3). Computer modeling using Molecular Operating Environment generated three-dimensional structures of TRPM3. Epiallopregnanolone sulfate (PM5S) concentrations were reduced in GDM (P < 0.05), in women with higher fasting plasma glucose (P < 0.010), and in early pregnancy samples from women who subsequently developed GDM with BMI ≥35 kg/m² (P < 0.05). In islets, 50 µmol/L PM5S increased GSIS by at least twofold (P < 0.001); isosakuranetin (TRPM3 inhibitor) abolished this effect. PM5S increased calcium influx in TRPM3-expressing HEK293 cells. Computer modeling and docking showed identical positioning of PM5S to the natural ligand in TRPM3. PM5S increases GSIS and is reduced in GDM serum. The activation of GSIS by PM5S is mediated by TRPM3 in both mouse and human islets.     

Illusory conjunctions in visual short‐term memory: Individual differences in corpus callosum connectivity and splitting attention between the two hemifields

Overloading the capacity of visual attention can result in mistakenly combining the various features of an object, that is, illusory conjunctions. We hypothesize that if the two hemispheres separately process visual information by splitting attention, connectivity of corpus callosum—a brain structure integrating the two hemispheres—would predict the degree of illusory conjunctions. In the current study, we assessed two types of illusory conjunctions using a memory‐scanning paradigm; the features were either presented across the two opposite hemifields or within the same hemifield. Four objects, each with two visual features, were briefly presented together followed by a probe‐recognition and a confidence rating for the recognition accuracy. MRI scans were also obtained. Results indicated that successful recollection during probe recognition was better for across hemifields conjunctions compared to within hemifield conjunctions, lending support to the bilateral advantage of the two hemispheres in visual short‐term memory. Age‐related differences regarding the underlying mechanisms of the bilateral advantage indicated greater reliance on recollection‐based processing in young and on familiarity‐based processing in old. Moreover, the integrity of the posterior corpus callosum was more predictive of opposite hemifield illusory conjunctions compared to within hemifield illusory conjunctions, even after controlling for age. That is, individuals with lesser posterior corpus callosum connectivity had better recognition for objects when their features were recombined from the opposite hemifields than from the same hemifield. This study is the first to investigate the role of the corpus callosum in splitting attention between versus within hemifields.     

Rat sperm immobilisation effects of a protein from Ricinus communis (Linn.): an in vitro comparative study with nonoxynol‐9

Previous study conducted in our department showed that 50% ethanolic extract of the root of Ricinus communis possess reversible antifertility effect and a 62‐kDa protein (Rp) from this extract is responsible for the antifertility effects. In this study, we compared the spermicidal effect of this Rp with nonoxynol‐9 (N‐9) in vitro. The sperm immobilisation studies showed that 100 μg ml^?1 of Rp was able to immobilise the sperms completely within 30 s. Sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo‐osmotic swelling in Rp and N‐9 treated groups in comparison with the control. In Rp and N‐9 treated groups, the number of acrosome‐reacted cells was found to be high and also caused agglutination of the spermatozoa, indicating the loss of intactness of the plasma membrane, which was further supported by the significant reduction in the activity of membrane bound 5′‐nucleotidase, acrosomal acrosin. In short, the protein Rp possesses spermicidal activity in vitro and its effects are similar to that of nonoxynol 9.     

Roles of Glycoproteins and Oligosaccharides Found in Human Vaginal Fluid in Bacterial Adherence

Adherence of type 1-piliated Escherichia coli to carbohydrate structures of vaginal mucosa plays a major role in the pathogenesis of ascending urinary tract infections in women. Colonization of the vaginal introitus is influenced by interactions between pathogens, vaginal fluid, and vaginal epithelium. In this study, the type and amount of carbohydrates and glycoproteins present in vaginal fluid were determined. Free and protein-bound oligosaccharides in vaginal fluid specimens were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-pressure liquid chromatography (HPLC). Two-dimensional electrophoretic separations of vaginal fluid glycoproteins were performed together with bacterial overlay assays. The results of FACE showed that the majority of the oligosaccharides are in the free state and the bound oligosaccharides are undetectable. HPLC analysis of free sugars revealed glucose as the major sugar (3.3 +/- 0.3 mM), and the concentrations of mannose and glucosamine were 0.065 +/- 0.04 and 0.02 +/- 0.001 mM, respectively. Radiolabeled E. coli bound three vaginal fluid glycoproteins with the following molecular masses and pIs: 82 kDa and pI 5.5, 55 kDa and pI 4.5, and 55 kDa and pI 6.5. The binding was inhibited by mannose and by deglycosylation of the proteins prior to the overlay assay. One of these putative receptors was identified to be the heavy chain of secretory IgA (S-IgA). These data suggest that the free mannose in the fluid is less than that required to affect E. coli-epithelial cell binding interactions and that S-IgA may bind E. coli in the vaginal introitus.