Genomic scan revealed KIT gene underlying white/gray plumage color in Chinese domestic geese.

Goose is an important type of domesticated poultry. The wild geese that are regarded as the ancestors of modern domestic geese present gray plumage. Domesticated, geese have both white and gray feathers. To elucidate the genetic mechanisms underlying the formation of white and gray plumage in geese, we resequenced the whole genome of 18 geese from six populations including white and gray goose breeds. The average sequencing depth per individual was 9.81× and the average genome coverage was 96.8%. A total of 346 genes were detected in the top 1% of FST scores of gray- and white-feathered geese, and a significant FST site was located in the intron region within the KIT gene, the 18 bp deletion in KIT having the strongest potential association with white feathers. It has been reported that a number of genes are associated with plumage colors in birds. However, no studies have identified the relationship between KIT and plumage color in birds at present, although the white coat can be attributed to mutations in KIT in some mammals. Our study showed that that KIT is a plausible candidate gene for white/gray plumage color in Chinese domestic geese.

[Discussion on stability of mixed standard application solution by volatilization organic compounds in workplace air].

Objective: To discuss the stability of mixed standard application solution by volatilization organic matter (benzene, toluene, xylene, ethylbenzene, acetone, butanone, Ethyl acetate, butyl ester, trichloroethylene) in workplace air. Methods: A total of 11 kinds volatilization organic compounds were separated by capillary chromatographic column, and detected with flame ionization detector. The stability of mixed standard application solution was judged by studying three paratemers during the placement period, such as the linear correlation coefficient of the standard curve, the relative response values of the highest and lowest concentration levels and the accuracy of the measured values of the quality control samples. Results: Within 187 days, the linear correlation coefficient of each compound was ≥0.999. The changes of relative response values at the highest and lowest concentration levels were both <10%; The measured values of toluene were all in the reference range. Conclusion: The mixed standard application solution is stable and reliable within 187 days of storage.

Higher thyroid-stimulating hormone levels in the first trimester are associated with gestational diabetes in a Chinese population.

AIM: To evaluate the relationship between maternal thyroid-stimulating hormone levels during the first trimester and gestational diabetes risk. METHODS: In Tianjin, China, 7258 women underwent a thyroid-stimulating hormone screening test within 12 gestational weeks and then had a glucose challenge test at 24-28 weeks of gestational age. The women with a glucose challenge test ≥7.8 mmol/l underwent a 75 g oral glucose tolerance test. Gestational diabetes was diagnosed following International Association of Diabetes and Pregnancy Study Group criteria. Restricted cubic spline analysis was performed to explore full-range risk associations of thyroid-stimulating hormone levels with gestational diabetes. Logistic regression was performed to obtain odds ratios and 95% confidence intervals. RESULTS: In all, 594 women (8.2%) had gestational diabetes. Among women with thyroid-stimulating hormone ≤3.2 mIU/l, a positive association between thyroid-stimulating hormone levels and gestational diabetes risk was found (adjusted OR: 1.13, 95% CI: 1.00-1.27). There was no relationship between thyroid-stimulating hormone levels and gestational diabetes risk in univariable and multivariable analyses among women with thyroid-stimulating hormone >3.2 mIU/l. In subgroup analyses, among women with thyroid-stimulating hormone ≤3.2 mIU/l and BMI ≥25 kg/m2 , the adjusted odds ratio for thyroid-stimulating hormone levels with gestational diabetes was enhanced to 1.25 (95% CI: 1.02-1.53). CONCLUSIONS: In pregnant Chinese women, thyroid-stimulating hormone levels even within normal range in the first trimester were positively related to gestational diabetes risk, especially for pre-pregnancy overweight/obese women.

Metformin treatment of antipsychotic-induced dyslipidemia: an analysis of two randomized, placebo-controlled trials.

Dyslipidemia is one of the most common adverse effects in schizophrenia patients treated with antipsychotics. However, there are no established effective treatments. In this study, data were pooled from two randomized, placebo-controlled trials, which were originally designed to examine the efficacy of metformin in treating antipsychotic-induced weight gain and other metabolic abnormalities. In total, 201 schizophrenia patients with dyslipidemia after being treated with an antipsychotic were assigned to take 1000 mg day-1 metformin (n=103) or placebo (n=98) for 24 weeks, with evaluation at baseline, week 12 and week 24. The primary outcome was the low-density lipoprotein cholesterol (LDL-C) levels. After metformin treatment, the mean difference in the LDL-C value between metformin treatment and placebo was from 0.16 mmol l-1 at baseline to -0.86 mmol l-1 at the end of week 24, decreased by 1.02 mmol l-1 (P<0.0001); and 25.3% of patients in the metformin group had LDL-C ≥3.37 mmol l-1, which is significantly <64.8% in the placebo group (P<0.001) at week 24. Compared with the placebo, metformin treatment also have a significant effect on reducing weight, body mass index, insulin, insulin resistance index, total cholesterol and triglyceride, and increasing high-density lipoprotein cholesterol. The treatment effects on weight and insulin resistance appeared at week 12 and further improved at week 24, but the effects on improving dyslipidemia only significantly occurred at the end of week 24. We found that metformin treatment was effective in improving antipsychotic-induced dyslipidemia and insulin resistance, and the effects improving antipsychotic-induced insulin resistance appeared earlier than the reducing dyslipidemia.

Structure-dependent behaviours of skin layers studied by atomic force microscopy.

The multilayer skin provides the physical resistance and strength against the environmental attacks, and consequently plays a significant role in maintaining the mammalian health. Currently, optical microscopy (OM) is the most common method for the research related to skin tissues while with the drawbacks including the possibility of changing the native morphology of the sample with the addition of the chemical or immunological staining and the restricted resolution of images for the direct observation of the tissue structures. To investigate if the function of each tissue is structure-dependent and the how the injured skin returns to the intact condition, we applied atomic force microscopy (AFM) on the sectioned mice-skin to reveal the tissue structures with a nanoscale resolution. From the outermost stratum to the inner layer of the skin tissue, the respectively laminated, fibrous, and brick-like structures were observed and corresponded to various functions. Due to the mechanical differences between the tissue constituents and their boundaries, the sizes and arrangements of the components were characterised and quantified by the mechanical mapping of AFM, which enabled the analytical comparisons between tissue layers. For the wound model, the skin tissues were examined with the initial formation of blood vessels and type-I collagen, which agreed with the stage of healing process estimated by OM but showed more detail information about the evolution of proteins among the skin. In conclusion, the characterisation of the components that consist of skin tissue by AFM enables the connection of the tissue function to the corresponded ultrastructure.

[Anatomy research of cervical laminoplasty with preservation of the posterior ligament complex.]

Objective: To evaluate the feasibility of cervical laminoplasty with preservation of the posterior ligament complex for enlarging the spinal canal. Methods: Six up-to-standard human corpse specimens were divided into two groups by simple randomization (start from C4 group, S4; start from C5 group, S5; 3 corpses in each group). Decompression operation of C3-C6 level was performed in a predetermined sequence by using the new procedure with preservation of the posterior ligament complex.The basic depth of spinal canal was measured with a depth gauge at fixed point after the right bone groove of single level was completed.The operation of the contralateral bone groove was continued, and then the spinal canal was measured again when the spinous process was pulled backward by using a tissue forceps until the ligament complex was just tight.Retreat value (RV) of vertebral lamina was obtained by calculating the difference between the two measurements.The earlier measured levels needed to be remeasured when the operation area increased by one level. Two independent sample and one-sample t test were used to analyze the measurement results. Results: RV of vertebral lamina was small after finishing the first level of the decompression operation [S4: (0.87±0.72) mm; S5: (1.83±0.29) mm], and the value reached its maximum after the completion of C3-C6 level.The overall average RVmaxs from C3 to C6 level were (2.37±0.52) mm, (4.27±0.78) mm, (3.73±0.93) mm and (2.16±0.77) mm, respectively.The overall average retreat rates (RR) were 17%±7%, 32%±9%, 29%±10% and 16%±6%, respectively. The overall average RVmax of C4 and C5 level reached or exceeded the decompression threshold value of 4 mm (t=0.839, -0.703, both P>0.05). The average RVmax of C4/C5 level was similar in the two groups (t=-1.204, 1.189, both P>0.05); however, the difference of average RVmax between C3 and C6 level was significant (t=-4.429, 4.196, both P<0.05). Conclusions: Cervical laminoplasty with preservation of the posterior ligament complex can enlarge the sagittal diameter of spinal canal and relieve the compression of spinal cord.In addition, RV of each level increases as the number of the operation level increases, and the ability of vertebral lamina to retreat is quite different from C3 to C6 level.The decompression effect in the middle of the operation area is better than that on the cranial and tail side.

Treatment of aging oily wastewater by demulsification/flocculation.

The aging oily wastewater (AOW) from Tarim oilfield in China was treated by demulsification/flocculation. A novel sewage treatment agent (YL-7) was developed using a cationic surfactant (LY) and flocculants (polydimethyl diallyl ammonium chloride (PDMDAAC)/polyaluminum chloride (PAC)). At an YL-7 dosage of 320 mg L(-1) at 323 K for 90 min, the oil content of AOW was reduced from 728.8 mg L(-1) to 23.7 mg L(-1), and oil removal efficiency reached 96.7%. Microorganism flocs (extracted from AOW) with high negative zeta potential enhanced the stability of oil/water emulsion. LY and PDMDAAC neutralized the negative charge on the oil droplet surface. PDMDAAC and PAC mainly bridged and swept flocs during the flocculation process. YL-7 was found to be a suitable sewage treatment agent in removing oil from AOW.

Microwave-assisted extraction and purification of chlorogenic acid from by-products of Eucommia Ulmoides Oliver and its potential anti-tumor activity.

An efficient method for the rapid extraction, separation and purification of chlorogenic acid (CGA) from by-products of Eucommia Ulmoides Oliver (E. ulmoides) by microwave-assisted extraction (MAE) coupled with high-speed counter-current chromatography (HSCCC) was developed. The optimal MAE parameters were evaluated by response surface methodology (RSM), and they were extraction time of 12 min, microwave power of 420 W, ethanol concentration of 75 %, solvent/sample ratio of 30:1 (mL/g), yield of CGA reached 3.59 %. The crude extract was separated and purified directly by HSCCC using ethyl acetate-butyl alcohol-water (3:1:4, v/v) as the two-phase solvent system. The 14.5 mg of CGA with the purity of 98.7 % was obtained in one-step separation from 400 mg of crude extract. The chemical structure of CGA was verified with IR, ESI-MS analysis. Meanwhile, the purified CGA extract was evaluated by MTT assay and results indicate that CGA extract exhibited potential anti-tumor activity for AGS gastric cancer cell.

Hybrid laparoscopic technique for renal artery Takayasu arteritis.

OBJECTIVE: To evaluate the feasibility of combined laparoscopic technique for different types of vascular reconstruction in the treatment of Takayasu renal artery stenosis. DESIGN: Retrospective study of seven cases of renal artery stenosis caused by Takayasu arteritis (TA). MATERIALS: Institutional practice and hospitalised patients. All these patients manifested renal arterial hypertension and failed to percutaneous transluminal angioplasty (PTA) treatment. Different types of revascularisation using hybrid laparoscopic technique were applied. METHODS: Laparoscopic renal artery isolation and kidney mobilisation were first performed. Several types of vascular reconstruction were performed as two patients underwent autotransplantation, four patients aortorenal bypass and one splenorenal bypass. For bypass patients, hypogastric artery was harvested by laparoscopic approach while saphenous vein and spleen artery were dissected by conventional opening. Autotransplantation and arterial anastomosis were then performed through an open incision. RESULTS: All procedures were performed successfully without major intraoperative complications. The total operative time was 191 (130-280) min while laparoscopic part was 62 (40-105) min. The mean blood loss was 261 (150-400) ml. Postoperative blood pressure returned to normal in five patients but two others required single-agent antihypertensive medication. Minor complications included lumbar artery injury and flank pain each in one case. The anastomosis was patent in all patients and no re-stenosis occurred during 6-40 months of follow-up. CONCLUSIONS: Hybrid laparoscopic techniques involving renal artery dissection and hypogastric artery harvesting are feasible in surgical treatment of Takayasu renal arteritis. This hybrid surgical technique provides an alternative approach to revascularise the renal circulation, especially for the patients of PTA treatment failure.

Mechanically controllable nonlinear dielectrics.

Strain-sensitive Ba x Sr1-x TiO3 perovskite systems are widely used because of their superior nonlinear dielectric behaviors. In this research, new heterostructures including paraelectric Ba0.5Sr0.5TiO3 (BSTO) and ferroelectric BaTiO3 (BTO) materials were epitaxially fabricated on flexible muscovite substrate. Through simple bending, the application of mechanical force can regulate the dielectric constant of BSTO from -77 to 36% and the channel current of BTO-based ferroelectric field effect transistor by two orders. The detailed mechanism was studied through the exploration of phase transition and determination of band structure. In addition, the phase-field simulations were implemented to provide theoretical support. This research opens a new avenue for mechanically controllable components based on high-quality oxide heteroepitaxy.

Development and Validation of a Fall Prevention Knowledge Test.

Falls are a serious, persistent problem in hospitals. Ensuring that all hospital staff have adequate knowledge of how to prevent falls is the first step in prevention. We identified validated fall prevention knowledge tests (FPKTs) and planned to conduct a systematic literature review. When the review identified a lack of FPKTs, we developed and evaluated a FPKT, confirmed its conceptual framework, identified the content domain, drafted test items, devised the format, selected items for empirical examination, and conducted a psychometric evaluation. We randomly divided a 209-subject data set into test and validation samples to make item reduction decisions and examine reliability and validity. The typical respondent was a white, 42-year old female nurse with a bachelor's degree and 7 years' experience. Subjects were confident in their ability to prevent falls, rating themselves an 8 on a self-efficacy scale of 1 (not at all) to 10 (very). The 11-item FPKT scale (range 0-11) attained a tetrachoric coefficient of 0.73, confirming initial reliability. FPKT mean scores obtained before and after fall prevention education improved from 5.1 ± 1.8 to 6.6 ± 1.7. Statistically significant differences (paired t-test = 12.4, p < .001) confirmed validity. A robust way to assess nurses' knowledge of fall prevention is needed to inform effective educational programs. Addressing gaps in validated FPKTs provides an opportunity to inform and evaluate effective fall prevention programs. J Am Geriatr Soc 67:133-138, 2019.

Insulin resistance in Alzheimer's disease (AD) mouse intestinal macrophages is mediated by activation of JNK.

OBJECTIVE: Alzheimer's disease (AD) has been considered as a metabolic disorder disease, which closely related to insulin signaling impairment. Therefore, identifying the potential mechanism of insulin resistance is important for AD treatment. MATERIALS AND METHODS: An APP/PS1 double transgenic AD mouse model was introduced to study insulin resistance in gut. The expressions of AD markers and key elements of insulin signaling were detected in ileum and intestinal macrophages of AD mice by immunohistochemistry. Furthermore, mouse intestinal macrophage cell line RAW264.7 was treated by Aß25-35 or Aß25-35 + insulin to explore the mechanism of insulin resistance in vitro. The expression of IR-ß and the activation of cell signaling related proteins (Insulin receptor substrate 1 (IRS1), protein kinase B (AKT) and c-Jun N-terminal kinase (JNK)) in Aß25-35-stimulated macrophages were performed via Western blotting. RESULTS: The expressions of IRS1, Aß and Tuj in AD mice ileum were significantly different from WT mice (p<0.05). Also, there were significant discrepancies in the expressions of ß2AR and eNOS in intestinal macrophages of two groups (p<0.05). After exposure to Aß25-35, cell proliferation rate (p<0.01) of macrophage and the levels of TNF-α (p<0.01) and Il-6 (p<0.01) was significant elevated and treatment with insulin could reverse these changes (p<0.05). The amount of IR-ß and the p-AKT/AKT ratio significantly decreased in Aß25-35-treated macrophages (p<0.01), while the ratios of p-IRS1/IRS1 and p-JNK/JNK significantly enlarged (p<0.01). Furthermore, all the changes caused by Aß25-35 treatment were attenuated by insulin addition. CONCLUSIONS: Activation of JNK pathway played an important role in insulin resistance of AD mice, suggesting that inhibition of JNK pathway might be a new strategy toward resolving insulin resistance related diseases, such as AD.

[Effect of inserted angle on the stability of loaded microscrews].

Objective: To evaluate the effect of different insertion angles on the osseointegration of loaded microscrews in beagle jaws. Methods: Forty-eight microscrews were inserted at four different angles (30°, 50°, 70° and 90°) into the interradicular zones between the mandibular first molar and third premolar in twelve beagles and the microscrews had been loaded with a force of 2 N immediately for 8 weeks. After microscrew-bone specimens fixed, the maximum output value (Fmax) of pull-out test was recorded and the histomorphological changes of hard tissue were observed. The bone-implant contact (BIC%) was quantitatively analyzed and the osseointegration of microscrew-bone interface was comprehensively evaluated. Results: Both Fmax and BIC% values of microscrews were influenced by the insertion angles. The maximum value of Fmax was (385±23) N in the group with 50° angle, and the minimum value was (198±16) N in the group with 30° angle(P <0.05). The maximum value of BIC% was (59.1±6.0)% in the group with 70° angle, and the minimum value was (30.2±3.2)% in the group with 30° angle (P <0.05). Histomorphology observation revealed that in peri-screws region, the various degree of bone remodeling was found in different angle samples. Conclusions: The insertion angles (50°and 70°) were favorable to the stability of the microscrew.

Expression and clinical significance of miR-181a and miR-203 in systemic lupus erythematosus patients.

OBJECTIVE: MiR-181a plays a critical role in modulating T cell and B cell differentiation, as well as immune response. Its abnormal expression probably participates in the pathogenesis of systemic lupus erythematosus (SLE). MiR-203 is involved in regulating Toll-like receptor and inducing immune tolerance. Abnormal expression or function of miR-203 is related to multiple auto-immune diseases but its role in SLE remains unclear. This study, thus, investigated the serum level of miR-181a and miR-203, to analyze their roles in diagnosing and evaluating SLE. PATIENTS AND METHODS: SLE patients were recruited from our hospital, and divided into non-active and active SLE based on disease activity index, along with healthy individuals. qRT-PCR was used to quantify the serum miR-181a and miR-203 expression, and their correlation with clinical features. ROC was used to evaluate the diagnostic value on SLE, while survival curves were compared to show progression-free survival (PFS) between populations with high and low expression. RESULTS: SLE patients had significantly higher serum levels of miR-181a and lower miR-203, both of which were correlated with SLE activity. Expression levels of miR-181a and miR-203 were correlated with erythrocyte sedimentation rate, C reactive protein, anti-dsDNA antibody, complements, and SLEDAI score. Their expression levels had certain values in the differential diagnosis for active SLE (AUC=0.885 and 0.843). PFS in miR-181a high-expression individuals was lower than that in the low-miR-181 group (χ2=7.474, p=0.029). Whilst, miR-203 high-expression SLE patients had higher PFS than low-expression group (χ2=4.367, p=0.037). CONCLUSIONS: SLE patients had higher miR-181a and lower miR-203 expression, which thus may have critical implications in disease diagnosis and evaluation.

Molecular genetic and chemical assessment of Rhizoma Curcumae in China.

Rhizoma Curcumae (Ezhu) is a traditional Chinese medicine that has been used in removing blood stasis and alleviating pain for over a thousand years. Three species of Curcuma rhizomes are being used, which include Curcuma wenyujin, Curcuma phaeocaulis, and Curcuma kwangsiensis. In China, the production of Rhizoma Curcumae largely depends on agricultural farming. The essential oils are considered as active constituents in Rhizoma Curcumae, which include curdione, curcumol, and germacrone. On the basis of the yield of curdione, curcumol, and germacrone in an orthogonal array design, the optimized extraction condition was developed. The amounts of these compounds within essential oils in Rhizoma Curcumae varied according to different species and their regions of cultivation. Chemical fingerprints were generated from different species of Curcuma, which therefore could serve as identification markers. In molecular genetic identification of Rhizoma Curcumae, the 5S-rRNA spacer domains of 5 Curcuma species, including the common adulterants of this herb, were amplified, and their nucleotide sequences were determined. Diversity in DNA sequences among various species was found in their 5S-rRNA spacer domains. Thus, the chemical fingerprint together with the genetic distinction could serve as markers for quality control of Curcuma species.

Mutagenicity of ZnO nanoparticles in mammalian cells: Role of physicochemical transformations under the aging process.

Zinc oxide nanoparticles (ZnO NPs) potentially undergo physicochemical transformation in the environment, which may lead to unexpected environmental and health risks. The "aging" process is essential for better understanding the toxicity and fate of NPs in the environment. However, the mutagenic effects of aged ZnO NPs are still unexplored. The present study focused on investigating the physicochemical transformation during aging process and clarifying the mutagenicity of naturally aged ZnO NPs in human-hamster hybrid (AL) cells. It was found that ZnO NPs underwent sophisticated physicochemical transformations with aging regardless of original morphology or size, such as the microstructural changes, the formation of hydrozincite (Zn5(CO3)2(OH)6) and the release of free zinc ions. Interestingly, the aged ZnO NPs were investigated to be able to result in much lower cytotoxicity while relatively high degree mutation than fresh ZnO NPs. With characterization of the soluble and insoluble fractions of aged ZnO NPs suspension, together with the control measurements using metal chelator (TPEN) and endocytosis inhibitor (Nystatin), it was revealed that the release of zinc ions and nanoparticle uptake made significantly different contributions to the mutagenicity of fresh and aged ZnO NPs. This study clearly demonstrated that the physicochemical transformation of ZnO NPs with aging plays important and comprehensive roles in the ZnO NPs-induced mutagenicity in mammalian cells.

Monitoring bone resorption in early postmenopausal women by an immunoassay for cross-linked collagen peptides in urine.

A new immunoassay using an ELISA approach for measuring urinary excretion of cross-linked N-telopeptides of type 1 collagen was evaluated as a specific measure of bone resorption. The assay was applied to 65 early postmenopausal women who participated in a placebo-controlled trial of the aminobisphosphonate, alendronate sodium. Eight blood and urine samples were collected over a 9 month interval. Baseline cross-linked peptide excretion varied from 26 to 216 pmol BCE (bone collagen/mumol Cr. Within-subject variability (CV) for cross-linked peptide excretion was 20.2% over the 9 months in placebo-treated subjects, substantially less than that observed for other biochemical markers of bone resorption: 45, 53, and 63% for fasting urinary calcium and hydroxyproline and 24 h urinary lysylpyridinoline (HPLC assay), respectively. Baseline cross-linked peptide excretion correlated significantly (p < 0.001) with baseline total urine lysylpyridinoline and serum osteocalcin, but not with the other biochemical markers. Initial peptide excretion also correlated inversely with lumbar spine bone mineral density at entry (r = -0.26, p < 0.05). Treatment for 6 weeks with alendronate produced a dose-dependent suppression of cross-linked peptide excretion (0 +/- 8, 29 +/- 6, 56 +/- 5, and 64 +/- 3% for 0, 5, 20, and 40 mg, respectively, p < 0.01 versus placebo for treatment effect), with a return toward pretreatment values during follow-up. Measurement of the urinary cross-linked N-telopeptides of type I collagen by this new ELISA approach appears promising as a simple and reliable method to assess overall bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of calcium supplementation on the circadian rhythm of bone resorption.

Bone resorption shows a circadian rhythm in human subjects, but the physiological mechanisms underlying this rhythm are unknown. We compared the circadian rhythm of bone collagen degradation in 18 premenopausal women before and after oral calcium supplementation (1000 mg calcium for 14 days). Subjects were randomized to receive calcium at either 0800 h or 2300 h. Continuous 48-h urine collections and 1 day of 4-h urine collections were obtained before and after the 14-day supplementation period. We measured urinary deoxypyridinoline (Dpd) and the cross-linked N-telopeptide of type I collagen (NTx) as biochemical markers of bone resorption. There was a significant effect of time of day on excretion of Dpd and NTx (analysis of variance, P < 0.001) with peak excretion between 0300-0700 h and a nadir between 1500-1900 h. The mean amplitude (peak to trough) was similar for Dpd and NTx (70.3% and 63.3%, respectively). Evening calcium supplementation resulted in marked suppression of the nocturnal increase in Dpd and NTx and reversed the usual nocturnal increase in the level of parathyroid hormone. In contrast, morning calcium supplementation had no significant effect on the circadian rhythm of Dpd or NTx. Evening calcium supplementation suppressed overall daily excretion of Dpd by 20.1% (P = 0.03) and NTx by 18.1% (P = 0.03). Morning calcium supplementation had no significant effect on overall daily excretion of either Dpd or NTx. We conclude that evening calcium supplementation suppresses the circadian rhythm of bone resorption. The daily rhythm of PTH secretion or calcium intake is likely to be an important determinant of this rhythm. Experimental protocols designed to investigate the effect of calcium supplementation on bone mineral density should take the timing of supplementation into account.