Abstract:Objective:To investigate the expression and possible function of RhoA in human male reproduction. Methods:RT-PCR was used to detect RhoA mRNA, Western blot and Immunohistochemistry were used to detect the RhoA protein expression in human testis,epididymis and mature spermatozoa. The localization of this protein on human spermatozoa was determined by indirect immunofluorescent staining using anti-RhoA antibody. The percentage of acrosome reaction was determined by PSA staining. Results:RhoA mRNA was only detected in the epididymis,but its protein could be expressed in mature sperm as well as in the epididymal epithelium. RhoA was localized on human sperm entire acrosome and the flagellum,and it was translocated to the equatorial regions and post-acrosomal region after acrosome reacted. Meanwhile,the percentage of acrosome reacted sperm was decreased in comparison to pre-immune control after treatment with specific anti-RhoA antibody(P < 0.01). Conclusion:The results suggest that RhoA might be absorbed or incorporated into the human sperm plasma membrane during epididymal transition and could help human sperm to develop the ability to undergo acrosome reaction.

Abstract:Objective:To explore the major factor of enhancing monocyte recruitment into the peritoneal cavity. Methods:The stromal cell of ectopic implants was exposed to various concentrations(5,10,20 ng/ml ) of IL-1β and the 10 ng/ml of IL-1β was cocultured with ectopic implants,eutopic and normal endometrium. Concentration of RANTES in the cell culture supernatant was measured with a sandwish ELISA. Results:The 10 ng/ml of IL-1β elicited the highest secretion of RANTES in ectopic implants. The RANTES secreted by the stromal cell of ectopic implants was increased more significantly than that in eutopic and normal endometrium(P < 0.01). After 60 h,the stromal cell of eutopic endometrium secreted much more RANTES than normal endometrium(P < 0.05). Conclusion:These findings are consistent with a feed-forward inflammatory loop among the activate macrophages, RANTES production by ectopic implants and further monocyte chemotaxis. We postulate that this pathway is activated in endometriosis, and the bioactivity of eutopic endometrium was different from normal endometrium.

Abstract:Objective:To establish a sandwich ELISA for quantitative measurement of ProMBP. Methods:Anti-ProMBP monoclonal antibodies(mAbs) prepared by our laboratory were purified by Protein A affinity chromatography and analyzed by SDS-PAGE and Western blotting to test their characteristics. All the mAbs were labeled with horseradish peroxidase by sodium oxidation method, and antibody pairs were performed using anti-ProMBP mAb as coating antibody and HRP labeled anti-ProMBP mAb as labeled antibody,which optimal concentrations were defined by titration. Standard curve was performed using purified PAPP-A/ProMBP tetramer and was judged by sensitivity,reproducibility and recovery rate. The ProMBP levels of sera were measured with this assay. Results:The optimal paired antibodies were anti-ProMBP mAb 4C6E5B9 and HRP labeled anti-ProMBP mAb 9G4A6G10 which optimal concentrations were 2.5 μg/ml and 1:3 000,respectively. The sensitivity of this assay was 2.0 ng/ml. The coefficients of variation were 4.6% to 6.2% within assay and 4.6% to 10.5% between assays. The recovery rate was 92% to 113%.The ProMBP levels of serum were(26.37 ± 2.90)ng/ml,(357.71 ± 33.60)ng/ml and(1088.77 ± 58.13)ng/ml, which were collected from normal non-pregnant women,9 to 11 weeks gestation and 15 to 23 weeks gestation respectively. Conclusion:A sandwich ELISA assay for detecting ProMBP was obtained successfully.

Abstract:Objective:To investigate the effect of brain derived neurotrophic factor on TrkB mRNA and Protein expression in embryonic rat brain suffered from intrauterine hypoxic-ischemic injury. Methods:The uterine arteries of pregnant rats were clamped for 30 min, and saline or BDNF was injected to the rat through caudal veins for 5 days. Rats with sham surgery were used as controls. The expression of TrkB mRNA and protein was measured using RT-PCR and Western blotting. Results:TrkB mRNA and levels of protein expression were higher in embryonic rat brain after clamping the uterine arteries of pregnant rats for 30 min than those in control group. BDNF injection could up-regulate the TrkB expression significantly. Conclusion:BDNF injection through rat caudal veins could up-regulate the levels of the TrkB mRNA and protein expression in embryonic rat brain suffered from intrauterine hypoxic-ischemic injury and may enhance its neuroprotection effect on cortical neurons.

Abstract:Objective:To evaluate the effect of genistein(Gen) on the meiotic maturation of mouse oocytes. Methods:Mice were treated with different concentrations of Gen by intraperitoneal injection,and the oocyte production,germinal vesicle breakdown (GVBD),the extruding of the first polar body(PB1),in vitro fertilization(IVF) of mouse oocytes and embryonic development were observed. Murine immature oocytes were cultured in the media containing different concentrations of Gen. The rates of GVBD and the extruding of PB1 of mouse oocytes were detected after culture in vitro for 16 h and 24 h,respectively. Results:Administration of various doses of Gen significantly increased the weight of uterine and the number of ovulations(except high-dose group),prevented GVBD and the extruding of PB1 of mouse oocytes in vitro(except Gen-0.5 group). At concentration of 50.0 mg/kg,the capacity of fertilization of mature oocytes following in vitro fertilization(IVF) and embryo developmental capacity were decreased. Gen inhibited GVBD and the extruding of PB1 of immature mice oocytes in a dose-dependent manner after culture in vitro. Conclusion:Gen can reversibly inhibit the maturation of murine immature oocytes. At high concentration,Gen affected the process of the meiotic maturation,decreased the capacity of fertilization in vitro,suggesting that excessive intake of Gen by women at childbearing age may lead to reduced fertility,or even cause sterility or infertility.

Abstract:Objective:To investigate the effects of Src signal pathway on the adhesion and migration in human breast cancer cells(MDA-MB-231) and its role in the reorganization of actin cytoskeleton. Methods:The MDA-MB-231 cells were treated with Src inhibitor PP2 in various concentrations(0.2,1.0,5.0 and 25 -滋mol/L) for 24 h. MTT assay was used to detect the adhesive ability of MDA-MB-231 cells to artificial basement membrane. Migration rate was measured by wound healing assay. To detect the reorganization of actin cytoskeleton,the contents of G-actin(in cytosolic fraction) and F-actin(in cytoskeletal fraction) in cells were evaluated by Western blot, and the distribution of F-actin stained by FITC-phalloidin in cells was also examined by fluorescence microscopy. Results:After treated with PP2 for 24 h,the ability of adhesion and migration in MDA-MB-231 cells were significantly reduced compared to the control cells in a dose-dependent manner. Consistently,the celluar F-actin was also depolymerized after PP2 treatment. Conclusion:Src inhibitor PP2 can inhibit the ability of adhesion and migration in MDA-MB-231 cells, which probably be through the reorganization of actin cytoskeleton.

Abstract:Objective:To determine the prevalence and characteristics of deafness-causing mutations in Connexin26(Cx26,GJB2)gene and mitochondrial DNA A1555G with non- syndromic hearing impairment(NSHI). Methods:The peripheral blood samples were obtained from the students of Nanjing Deaf-Blind School and healthy people in Jiangsu province. Using polymerase chain reaction (PCR),the code region of mitochondrial DNA and GJB2 gene were amplified. The GJB2 235delC mutation and mtDNA A1555G mutation was distinguished by restricted enzyme digestion. All of the subjects were sequenced. Results:Fifteen kinds of polymorphism about GJB2 gene,were found in 162 cases of controls and 9 kinds of pathologic mutations of GJB2 gene(V27I,E114G,I203T,V37I,176-191del16,235delC,299-300delAT,T123N and 504insGCAA) were identified in 135 patients. GJB2 235delC is the main pathogenic mutation site in GJB2 gene,which was found in 37cases(18 cases were homozygosis,19 cases were heterozygosis). The mitochondrial DNA A1555G mutation was not found in those subjects. Conclusion:The mutation of Cx26 genes is much responsible for non-syndromic hearing impairment in this region. Our data support the view that specific combinations of GJB2 mutation exist in different populations. 235delC is the main pathogenic mutation site in GJB2 gene in Chinese. The incidence of mtDNA 12SrRNA A1555G mutation in the deaf population of Nanjing,Jiangsu Province is lower than the average level of the general Chinese deaf population.

Abstract:Objective:To research the location and morphology of zebrafish tooth in adult stage. Methods:The zebrafish(ages greater than 3 months) were included in this experiment. The pharyngeal arch skeleton was dissected from the adult zebrafish. The morphology of tooth was observed under the stereomicroscope or under the light microscope by paraffinum section and HE staining. Results:There are several teeth located on the paired fifth pharyngeal arch. The teeth are acrodont that have no dental root. The shape of the teeth is triangulum whose tip is the teeth cusp. The left and right teeth cusp oppose to each other forward to the pharyngeal cavity. By HE staining,the histological character of teeth are calcified tissue as same as the dentin of human tooth. Conclusion:The future research about the morphology and development process of the zebrafish tooth will help to find a kind of optimal model animal to the tooth develpoment research.

Abstract:Objective:To ensure the course of the supratrochlear artery by studying the anatomy to provide anatomic basis for the design of the modified paramedian forehead flap. Methods:Seven cadavers(twelve supratrochlear arteries) were used in the study. The initial position, the course of the supratrochlear artery(supraorbital rim and midline as landmark) and the communication between the supratrochlear artery and other arteries were recorded. Results:The supratrochlear artery exited the foramen supratrochlear which was(1.33 ± 0.14) cm away from the midline and ascended inward. It had an angulus(about 82 degree) towards the supraorbital rim level. The course of the supratrochlear artery was divided into three parts:part I,the artery was located between the corrugator and orbicularis oculi muscles;partⅡ,close to the previous one,the artery piercing the frontalis muscle approximately at the supraorbital rim level and ascended on the surface of the frontalis muscle from the supraorbital rim level to the place(0.93 ± 0.23)cm above it;and part Ⅲ,it ascended continuely within the subcutaneous plane and then intracutaneous plane. The branch of the supratrochlear artery was found at the level(1.26±0.20)cm above the supraorbital rim in five cadavers(eight supratrochlear arteries). And it ascended outward within the subcutaneous plane. The supratrochlear artery communicated with the branch of the supraorbital artery and the frontal branch of the superficial temporal artery. The forehead arterial anastomotic plexus was organized by those arteries. No muscular branches was found in the frontalis muscle. Bilateral supratrochlear arteries communicated with each other. Conclusion:The supratrochlear artery travels superficial to the frontalis muscle after it reaches the supraorbital rim. So we can design the paramedian forehead flap by the course of the vessel, and the flap can be elevated without muscle,except for the pedicled part.

Abstract:Objective:To investigate the expression of BDNF and axon guidanc cue slit-2 after the spinal cord injury,simultaneously observe the ethology, morphology and SEP changes of rats. Methods:Male Sprague-Dawley rats were randomly divided into normal group,the sham-operation group and the operation group(1,3,5,7,14,21,28 days sub groups), each group with 5 rats. The expression of BDNF and slit-2 were detected through immunohistochemistry methods. The positive cells were analyzed by a computer image analysis system. Function changes were evaluated by BBB method, the morphological changes were obsered by HE staining, and the SEP change was also analyzed. Results:The limb function of experimental rats significantly recovered from 7 d to 28 d. The immunohistochemistry results indicated that expression of BDNF\slit-2 in the angulus anterior was low in either normal or sham operation group,while BDNF\slit-2 expressed actively at 24 h in the operation group,reached the peak at 3 days after operation. The morphology gradually changed from local hematoma to tissue degeneration. Conclusion:With the spinal cord injury of adult rats, BDNF and slit-2 increased with regularity, indicating the correlation between the two factors and the neuranagenesis. BDNF and slit-2 were mainly expressed in the ventricolumna,implying they might be correlated with the limb movement function restore.

Abstract:Objective:To investigate the mechanism of Pioglitazone(PGZ) on insulin resistance and type 2 diabetes mellitus by using the DIO(diet-induced obese) mice model. Methods:The C57BL/6J mice were randomly divided into the normal diet group and high-fat diet(HFD) group. After 20 weeks,the obese mice were randomly divided into obese control group,PGZ group. They were orally administered placebo and PGZ[10 mg/(kg·d)] respectively for two weeks. The adiponectin mRNA expression levels from adipose tissue were analyzed by real-time quantitative PCR. The levels of plasma glucose,serum insulin and adiponectin were measured by Biochemistry technology,Radioimmunity and ELISA. The adipocyte sizes were also observed by Immunohistochemistry. Results:The weight,plasma glucose and serum insulin levels increased(P < 0.05) in HFD group,and the adipocyte sizes were bigger. Comparing to obese controls,serum insulin and glucose levels significantly decreased(P < 0.05) and the adipocyte sizes reduced in PGZ treatment group, while plasma adiponectin level raised(P < 0.01). Moreover,the mRNA expressions of adiponectin upregulated(P < 0.05). Conclusion:The effects of PPAR-γ agonist on insulin resistance can be carried out through regulating the expression of adipocytokines.

Abstract:Objective:To study the existence status of the integron and transposable element of multi-resistant Acinetobactor baumannii isolates in clinic in Nanjing. Methods:The samples of 20 multi-resistant acinetobacter baumannii isolates were collected from Jul, 2007 to Oct, 2007 of patients in hospitals of Nanjing. To determine the sensitivity to the 11 antibacterial a broth induction method was used, the genetic marker of integron qacE△1-sul1 and transposable element tnpU were analyzed by polymerase chain reaction(PCR). Results:In the 20 AB isolates, the rate of the positive of integron qacE△1-sull is 70%, and the rate of transposable element tnpU is 65%. Conclusion:The positive rate of the integron qacE△1-sul1 and transposable element tnpU for multi-resistant acinetobactor baumannii is high in Nanjing region. It should be reevaluated the chlorhexidine used as precaution infection drug after operation.

Abstract:Objective:To investigate the protective effect of Edaravone against hepatic ischemia-reperfusion injury in rat and clarify its possible mechanism. Methods:The model of 70% hepatic ischemia-reperfusion was established in SD male rat. Twenty four healthy rats were randomly divided into 3 groups,S: sham operation group(n = 8),IR:ischemia-reperfusion group(n = 8),ED:Edaravone treatment group(n = 8). After 2 hours of reperfusion,the enzyme levels in serum were examined including ALT and AST. The SOD,MDA and MPO in liver tissues were measured. The expression of NF-κB in liver cell was determined in immunohistochemistry, and histologic changes was observed in HE staining. Results:After ischemia-reperfusion,the levels of ALT,AST,MDA, the activity of MPO and NF-κB expression in IR group was significantly higher than those in S group. The injury of liver tissue in IR group was more obviously,but the activity of SOD was decreased. After treatment of Edaravone,the all above abnormal parameters were retrieved remarkably(P < 0.01). Conclusion:This study suggests that Edaravone has a protective effect on liver against ischemia-reperfusion injury in rat. The mechanism of protection may be related to its scavenging of radical,inhibiting of lipid peroxidation,and suppressing of NF-κB activation.

Abstract:Objective:To investigate the protective effect of rhKD/APP on liver ischemia-reperfusion injury of rats and its mechanism. Methods:All rats were divided into 6 groups randomly, sham-operation group, model group, rhKD/APP treatment groups and Aprotinin group, 10 in each group. rhKD/APP was given with different dose in rhKD/APP treatment groups. Pathological model of liver ischemia-reperfusion injury of rats was set up. Rats were killed after 40 min ischemia and 3 h reperfusion. The levels of ALT, AST and ALP in serum were measured. Hepatic tissue was observed under light and electronic microscopy. The expression of TNF-α protein in tissue was determined by immunohistochemistry. Results:rhKD/APP can significantly decrease the activities of ALT, AST and ALP. Under light and electronic microscopy, the extent of damaged hepatic tissue were observed decreased in the groups treated with rhKD/APP. Expression of TNF-α protein in tissue was significantly reduced(P < 0.01). Conclusion:rhKD/APP has a protective effect on liver ischemia-reperfusion injury of rats. rhKD/APP can obviously inhibit the inflammatory process by inhibiting TNF-α.

Abstract:Objective:To investigate the effect on invasion and metastasis of HCCLM3 cells blocked peroxisome proliferators-activated receptor gamma(PPARγ) expression by RNA interference method. Methods:Transwell chambers were used to construct tumor cell invasive models,PPARγ shRNA(pshPPARγ group) and plasmid pBi-hU6-DNA(pshPPARγ-N group) were transfected into HCCLM3 cells. Untreated HCCLM3 cells were regarded as negative control(control group). The changes of PPARγ expression in three groups were detected by semi-quantitive RT-PCR and Western blot analysis. Cell adhesion,migration,invasion and locomotion of cells were observed,Meanwhile,MMP2,TIMP2 and integrin β1 mRNA expression were detected by RT-PCR. Results:PPARγ expression in pshPPARγ group was 80.5% lower than the other two groups at mRNA level. In pshPPARγ groups,the number of cells crawled over semipermeable membrane of Tanswell chambers were 68 ± 7,the number of cells permeated through ECM were 17 ± 3,and the ability of cell adhesion decreased significantly. After 2 h,adhesion cell counts in pshPPARγ groups are 49 ± 9,the time when cells healed the wound were(6.40 ± 1.14)d. There were significant differences between pshPPARγ group and the other two groups(P < 0.01). In pshPPARγ group,MMP2 and integrin β1 mRNA of HCCLM3 cells decreased significantly,but TIMP2 increased. Conclusion:Knockdown PPARγ expression with RNA interference technology can significantly inhibit the invasion and metastasis of HCCLM3 cells, which might be the result of the decrease expression of integrin β1 to balance MMP2/TIMP2.

Abstract:Objective:To determine the simple effect and interaction of parathyroid hormone(PTH) and 1,25(OH)2D3 on the regulation of renal calcium binding proteins. Methods:The expression of renal calbindin D28K and D9K were examined in mice with targeted knockout of the PTH or 1α(OH)ase or both genes by immunohistochemistry. Results:The calbindin D28K and D9K were localized to intracytoplasm of epithelia in the renal cortical distal tubules and medullary collecting ducts. The expression of calbindin D28K and D9K were reduced significantly in kidney in PTH knockout mice, more dramatically decreased in 1α(OH)ase knockout mice and the most dramatically decreased in the double mutant mice. Conclusion:PTH and 1,25(OH)2D3 could play collaborative roles in modulating the expression of calbindin D28K and D9K in the kidney.

Abstract:Objective:To study the effects of the lycopene on antioxidation in SD old rats. Methods:According to the initial contents of malondiadehycle(MDA) in the serum, 48 SD old rats were devided radomly to 4 groups. After the rats were orally administered with 0.066 4 g/(kg·d), 0.134 g/(kg·d), 0.4 g/(kg·d) lycopene and the oil for 30 days, the concents of serum MDA, the energy of serum SA, the energy of serum GSH-Px and the contents of lipofasciin in liver were observed. Results:Compared with the oil group, the concents of serum MDA were decreased significantly in 0.4 g/(kg·d) group, and the energy of serum SA was increased significantly in 0.134 g/(kg·d) and 0.4 g/(kg·d) groups. Conclusion:Lycopene has antioxidant effect on SD old rats.

Abstract:Objective:To study the effect of triptolide on the expression of VEGF and MMP-9 in the rheumatoid arthritis fibroblast-like synoviocyte line,MH7A. Methods:Rheumatoid arthritis fibroblast-like synoviocyte line,MH7A were incubated for 24 hours with 20 ng/ml of recombinant human TNF-α then were treated with Triptolide of different concentration(1 μg/ml,5 μg/ml,25 μg/ml and 75 μg/ml). Levels of vascular endothelial growth factor(VEGF) and matrix metallproteinases(MMP-9)were measured in cell supernatants by Enzyme-Linked ImmunoSorbent Assay(ELISA). Results:After TNFα stimulation,VEGF and MMP-9 production was highly increased in the supernatants. Triptolide caused a dose-dependent decrease in VEGF and MMP-9. The inhibited effect of Triptolide on the expression of VEGF and MMP-9 is stronger than that of Dexamethasone. Conclusion:Triptolide can inhibit the regulative factor of angiogenesis VEGF and MMP-9 expression.

Abstract:Objective:To determine the acceptability and influencing factors on HIV voluntary counseling and testing(VCT) among high risk population,proposing reasonable strategies on active seeking of VCT service. Methods:Quantitative questionnaire survey and qualitative individual interviews were conducted in the population with high risk behaviors in Nanjing,Nantong,Xuzhou city,and so on. Results:Total number of 1 721 objects covering five different high risk groups were recruited,only 31.8% of which had ever received VCT service. The main factors influencing VCT acceptability were as follows:low knowledge of VCT and low awareness on HIV testing. Among subjects never seeking VCT,40.2% people had never heard of VCT,and 49.7% thought it was unnecessary to access; Factors related to VCT services,76.8% reported preferred that the place for VCT was Centers for Disease Control and Prevention. 87.1% subjects hoped to obtain free AIDS counseling and testing delivered by a counselor in a separate room. The alternative way was free collective VCT in the entertainment establishments provided by team workers. Confidentiality and laboratory factors; about 44.7% and 43.2% of subjects were reluctant to seek VCT for being afraid of the poor confidentiality in the clinic or inaccurate laboratory test, respectively. Conclusion:Widely education programs on promoting VCT and demand were needed. High standard VCT service delivery and measures improvement can allure more people seeking VCT and make VCT service functional.

Abstract:Objective:To investigate the effect of aspirin on the function of endothelium-type nitric oxide synthase(eNOS) in platelets of type 2 diabetic patients. Methods:Platelets,isolated from blood of healthy human subjects and type 2 diabetic patients,were exposed in vitro to aspirin at three different concentrations(25,50,100 μmol/L) and vehicle respectively. Platelets aggregation was measured turbidimetrically with a NBC Hema Tracer 601 aggregometer. In platelets,eNOS activity was determined by the ratio of the conversion of L-[3H]-arginine to L-[3H]-citrulline. Intraplatelet cGMP,an index of bioactive NO,was measured by radioimmunoassay. Results:Compared with the vehicle,aspirin concentration-dependently decreased the platelets aggregation,increased the activity of eNOS in platelets and intraplatelet cGMP level significantly,both in healthy human control and type 2 diabetic patients. And the effect was greater in healthy human subjects than that in type 2 diabetic patients at the same concentration of aspirin. Conclusion:Aspirin can improve the function of the platelet eNOS in type 2 diabetic patients in a concentration-dependent manner.

Abstract:Objective:To investigate the serum levels of lipoxin A4(LXA4) in children with Henoch-Schonlein purpura(HSP) in different phase, and explore the correlation of LXA4 with interleukin(IL)-5,IL-6,IL-8 and CRP in the pathogenesis of HSP. Methods:The blood levels of LXA4,IL-5,IL-6,IL-8 and CRP in 26 normal children and 27 children with HSP in acute phase and earlier recovery phase were detected by ELISA,respectively. Results:The blood levels of LXA4,IL-5,IL-6,IL-8 and CRP in children with HSP in acute and earlier recovery phase were significantly higher than those in normal group(P < 0.01),and the blood level of LXA4 in children with HSP in earlier recovery phase was significantly higher than that in the acute phase(P < 0.01). However,the blood levels of IL-5,IL-6,IL-8 and CRP in children with HSP in acute phase were significantly higher than those in earlier recovery phase(P < 0.01). There were positive correlations between LXA4 and IL-5 or IL-8. Meanwhile,there were no positive correlations between LXA4 and IL-6,LXA4 and CRP. There were positive correlations among IL-5,IL-6,IL-8 and CRP. The LXA4 levels of severe patients in acute and earlier recovery phase were lower than that in mild cases. Conclusion:These data suggest that IL-5,IL-6 and IL-8 may play an injurious role, and LXA4 may play a protective role in the pathogenesis of HSP. The insufficiency of LXA4,an anti-inflammatory mediator in human body,may be a reason for the patients with HSP whose illness become more serious.

Abstract:Objective:To observe the effect of early nutrition in very low birth weight infants. Methods:Sixty-three preterm infants with birth weights <1 500 g received parenteral nutrition containing both 2.0 g/(kg·d) amino acids(AA) and 1.5 g/(kg·d) lipid within the first 24 hours with a increase of 1.0 g/(kg·d) in both of them(ETPN group), or solely glucose during the first day with 0.5 g/(kg·d) AA in second day and 0.5 g/(kg·d) lipid in the third day with a increase of 0.5 g/(kg·d) in both of them(ETPN group). The final dose of AA is 3.5 g/(kg·d) and lipid is 3.0 g/(kg·d). To compare nitrogen balances and caloric intake in the two groups,the plasma concentration of cholesterol,triglyceride,bilirubin,creatinine were determined to evaluate the side effect of ETPN. Results:VLBW infants supplemented with ETPN had no major adverse side effects. They got a positive nitrogen balance at the first day and more calories intake. Conclusion:Early total parenteral nutrition administration to VLBW infants results in a positive nitrogen balance and more calories intake.