The Unmixing Problem: A Guide to Applying Single-Cell RNA Sequencing to Bone.

Bone is composed of a complex mixture of many dynamic cell types. Flow cytometry and in vivo lineage tracing have offered early progress toward deconvoluting this heterogeneous mixture of cells into functionally well-defined populations suitable for further studies. Single-cell sequencing is poised as a key complementary technique to better understand the cellular basis of bone metabolism and development. However, single-cell sequencing approaches still have important limitations, including transcriptional effects of cell isolation and sparse sampling of the transcriptome, that must be considered during experimental design and analysis to harness the power of this approach. Accounting for these limitations requires a deep knowledge of the tissue under study. Therefore, with the emergence of accessible tools for conducting and analyzing single-cell RNA sequencing (scRNA-seq) experiments, bone biologists will be ideal leaders in the application of scRNA-seq to the skeleton. Here we provide an overview of the steps involved with a single-cell sequencing analysis of bone, focusing on practical considerations needed for a successful study. © 2019 American Society for Bone and Mineral Research.

Comparison of the effectiveness of medicinal leech and TENS therapy in the treatment of primary osteoarthritis of the knee : A randomized controlled trial.

OBJECTIVES: The aim of this study was to evaluate the effects of leech therapy in the treatment of knee osteoarthritis in terms of duration of effectiveness and symptom relief and to compare these results with transcutaneous electrical nerve stimulation (TENS) therapy. MATERIAL AND METHODS: This study was designed as a prospective, single center, randomized, single-blind and parallel group study. A total of 90 patients were included in the study, 46 in the leech group and 44 in the TENS group. Primary outcome measures were changes of the pain scores in visual analogue scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) on the measurements day 0, 21 and 180. Secondary outcome measures were the changes in the sub-groups of the WOMAC scores. Five leeches were applied to the affected knee, once every week for 3 weeks. RESULTS: The VAS pain score showed a similar decrease in both groups in the evaluation on day 21 (p < 0.001). The course of the change of the VAS pain score in both groups was similar in the comparisons between groups. Long-term benefits of the TENS therapy group were slightly more than the leech therapy group. All the sub-scores of WOMAC in both therapy groups showed a similar decrease (p = 0.819). Throughout the study this decrease was statistically significant in both groups (p < 0.001). CONCLUSION: Leech therapy relieves symptoms in patients with osteoarthritis of the knee and is as effective as TENS therapy in the management of osteoarthritis of the knee. This treatment has the potential of being an additional or alternative therapy for the non-surgical management of osteoarthritis of the knee.

Vegetable oils used as vitamin E vehicle affect the electrical activity of the rat heart.

The aim of this study is to define the possible effects of vegetable oils used as vitamin E vehicle on the electrical activity of the rat heart. To test the possible effects of vitamin E vehicles we studied the effect of i.p. injected corn oil, hazelnut oil or peanut oil on the action potential parameters recorded in both papillary and left atrial muscle strips. Four experimental groups were used. The control group was injected (i.p.) with distilled water, while the three remaining groups received injections of corn oil, hazelnut oil, or peanut oil for five weeks (in a dose of 0.4 ml/kg/day--minimum amount of oil in which vitamin E could be dissolved). We used borosilicated (15-20 megaohms) capillary electrodes and intracellular action potentials (AP) were recorded in isolated papillary and left atrium muscle strips. While administration of three different types of vegetable oil had no significant effect on AP parameters of papillary muscle, they significantly prolonged the repolarization phase of AP in atrial strips. These results show that vegetable oils used as vitamin E vehicles may alter the electrical activity of the heart in a tissue-dependent manner. The present data indicate that the possible effect of vegetable oil vehicles should be kept in mind while evaluating the possible effects of in vivo vitamin E administration.

A partial supplementation of pasteurized milk with vitamin C, iron and zinc.

After supplementation trials, vitamin C, iron and zinc levels were increased by 1789%, 59% and 30%, respectively. Partially supplemented pasteurized milk could be a new alternative with its high nutritive value, good sensory properties and low cost.

Dietary selenium and vitamin E intakes alter beta-adrenergic response of L-type Ca-current and beta-adrenoceptor-adenylate cyclase coupling in rat heart.

Previously we have shown that both insufficient (combined with vitamin E deficiency) and excess intake of selenium (Se) impairs isoproterenol (ISO)-induced contractions of rat papillary muscle. In the present study, we used patch-clamp and biochemical techniques to investigate mechanisms of this effect in rats fed a Se- and vitamin E-deficient, a Se-excess or a normal diet. Whole-cell configuration of patch-clamp technique was used to investigate L-type Ca(2+) currents (I(Ca,L)) and their regulation by beta-adrenergic receptor stimulation in enzymatically isolated single rat ventricular myocytes. Alteration of Se and vitamin E intake did not affect peak I(Ca,L), but the threshold potential of activation was significantly different among groups. Maximal I(Ca,L) responses to ISO were depressed in both experimental groups, but the EC(50) values were not affected. In the Se-deficient group, basal, ISO- or forskolin-induced adenylate cyclase (AC) activity, measured in cardiac membrane preparations, was reduced when compared to the control, whereas 5' guanylyimidodphosphate (GppNHp) stimulated activity was unaffected. Decreased beta-adrenoceptor density and reduced GppNHp-induced affinity shift in ISO binding were also observed in the deficient group. No such differences were present in the excess group. These results suggest that combined Se and vitamin E deficiency interferes with beta-adrenoceptor-AC coupling, whereas excess intake of Se does not affect it. Thus, in the deficient group, the impairment of I(Ca) responses to ISO may be a result of a defect in beta-adrenoceptor-AC pathway. Impairment of I(Ca) response in the excess group, however, appears to have a different underlying mechanism.

P2X7 purinoceptor expression in Xenopus oocytes is not sufficient to produce a pore-forming P2Z-like phenotype.

The purinergic rP2X7 receptor expressed in a number of heterologous systems not only functions as a cation channel but also gives rise to a P2Z-like response, i.e. a reversible membrane permeabilization that allows the passage of molecules with molecular masses of > or = 300 Da. We investigated the properties of rP2X7 receptors expressed in Xenopus oocytes. In two-electrode voltage-clamp experiments, ATP or BzATP caused inward currents that were abolished or greatly diminished when NMDG+ or choline replaced Na+ as the principal external cation. In fluorescent dye experiments, BzATP application did not result in entry of the fluorophore YO-PRO-1(2+). Thus, rP2X7 expression in Xenopus oocytes does not by itself give rise to the pore-forming P2Z phenotype, suggesting that ancillary factors are involved.

Expectant management of functional ovarian cysts: an alternative to hormonal therapy.

OBJECTIVE: We studied whether the administration of oral contraceptives facilitates the disappearance of spontaneously formed functional ovarian cysts. METHODS: Eighty patients in whom ultrasonography revealed unilateral, mobile, unilocular, thin-walled ovarian cysts without internal echoes and greater than 30 mm but not exceeding 60 mm in diameter were enrolled into the study. Patients were randomized by stratification to receive a low-dose monophasic pill, a high-dose monophasic pill, a multiphasic pill or no therapy. Patients were re-evaluated after 5 weeks of therapy and at the end of therapy (10 weeks). RESULTS: The mean ages and the mean cyst diameters of the patients for each group were not significantly different. We did not find a significant effect of oral contraceptive administration on the disappearance rate of functional ovarian cysts over that of expectant management. CONCLUSION: We conclude that oral contraceptive therapy even with multiphasic pills is very effective in the management of functional ovarian cysts but expectant management achieves similar success rates and may be a good alternative to oral contraceptive therapy.