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A study on gelatin capsule brittleness: moisture tranfer between the capsule shell and its content.

Variation in moisture content of the capsule shells either due to the change of storage conditions or the moisture transfer between the capsule shell and its contents may lead to undesired physical properties, such as capsule brittleness and stickiness. DMP 504, a developmental bile-acid sequestrant, is a strongly basic anion-exchange polymer which contains randomly distributed primary, secondary, tertiary, and quaternary amine groups in their hydrochoride salt form. The alkylammonium groups which comprise this polymer form a random network containing a high level of branching and a low level of cross-linking. DMP 504 is very hygroscopic and has a tendency to gain or lose moisture with ease. The transfer of moisture from the capsule shell to DMP 504 powder contained in a hard gelatin capsule can be expected, and if a low water content of the capsule shell is achieved, the capsules become brittle and fracture easily. The sorption isotherm for DMP 504 was generated by storing the drug substance under various relative humidity conditions. After equilibrium, the moisture contents for the samples of individual isotherm points were measured by thermogravimetric analyses. This report applies the sorption-desorption moisture transfer (SDMT) model to predict the equilibrium relative humidity in a system containing DMP 504 in hard gelatin capsules and to establish target loss on drying values for DMP 504 and the capsule shell. Application of this SDMT model resulted in finding a solution to the brittleness problem. The moisture levels of capsule shells and contents for two formulations in a 12-month stability program are also reported here. Results of this study further demonstrate that the SDMT model can be used as a tool to guide the formulator to select optimal initial moisture contents for the empty capsule shell and the formulation to avoid the incidence of brittle capsule problems.

Absorption↗

Electrostatic aggregation and formation of core-shell suprastructures in binary mixtures of charged metal nanoparticles.

Electrostatic aggregation of oppositely charged silver and gold nanoparticles leads to the formation of core-shell clusters in which the shell is formed by the nanoparticles, which are in excess. Arguments based on Debye screening of interactions between like-charged particles help explain why these clusters are stable despite possessing net electric charge. The core-shell aggregates exhibit unusual optical properties with the resonance absorption of the shell particles enhanced by the particles in the core and that of the core suppressed by the shell. Experimental UV-vis absorption spectra are faithfully reproduced by Mie theory. The modeling allows for estimation of the numbers of particles forming the shell and of the shell's effective thickness. These theoretical predictions are substantiated by experiments using nanoparticles covered with different combinations of charged groups and performed at different values of pH.

Complex Mixtures↗

Polymorphism of turnip yellow mosaic virus empty shells and evidence for conformational changes occurring after release of the viral RNA. A differential scanning calorimetric study.

Turnip yellow mosaic virus (TYMV) is a small isometric plant virus which decapsidates by releasing its RNA through a hole in the capsid, leaving behind an empty shell [R. E. F. Matthews and J. Witz, (1985) Virology 144, 318-327]. Similar empty shells (artificial top component, ATC) can be obtained by submitting the virions to various treatments in vitro. We have used differential scanning calorimetry, analytical sedimentation, and electron microscopy to investigate the thermodenaturation of natural empty shells (NTC, natural top component) present in purified virus suspensions, and of several types of ATCs. ATCs divided in two major classes. Those obtained by alkaline titration, by the action of urea or butanol behaved as NTC: their thermograms contained only one peak corresponding to the irreversible dissociation of the shells and the denaturation of the coat protein. The temperature of this unique transition varied significantly with pH, from 71 degrees C at pH 4.5 to 84 degrees C at pH 8.5. The thermograms of ATCs obtained by freezing and thawing, or by the action of high pressure, contained two peaks: shells dissociated first into smaller protein aggregates at 57 degrees C (at pH 5.0) to 61 degrees C (at pH 8.5), which denatured at the temperature of the unique transition of NTC. Shells obtained by heating virions to 55 degrees C at pH 7.6, changed conformation after the release of the viral RNA, as upon continuous heating to 95 degrees C, their thermograms were similar to those of the shells obtained by freezing and thawing, whereas after purification they behaved like NTC. Structural implications of these observations are discussed.

Calorimetry, Differential Scanning↗

Neither ibotenic acid nor volkensin lesions of the nucleus accumbens shell affect the expression of cocaine sensitization.

Studies have shown that the nucleus accumbens shell plays an integral role in the expression of psychostimulant-induced behavioural sensitization. Dopaminergic regulation of excitatory amino acid inputs in this region of the brain could be a key factor in the neural influence of this phenomenon. Alterations in the dopaminergic innervation patterns in the shell have been demonstrated in rats that received repeated cocaine injections. Furthermore, lesions of brain regions that send projections to the shell alter psychostimulant-induced locomotion, both acutely and in sensitization paradigms. A previous study from our laboratory demonstrated that lesions of the shell before repeated cocaine treatment decrease the locomotor response to cocaine during the induction phase of behavioural sensitization. To better understand the role of this brain region during the expression phase of behavioural sensitization, the present study examined the effects of two forms of cytotoxic lesions of the shell. Rats received a sensitization-inducing regimen of cocaine (bi-daily injections of 15 mg/kg i.p. for 5 consecutive days). Two days after the last injection, rats demonstrating behavioural sensitization received one of three bilateral microinjections into the shell: (i) 0.5 micro L 0.9% saline; (ii) 2.5 micro g/0.5 micro L ibotenic acid (which lesions the cell bodies at the injection site); or (iii), 0.5 ng/0.2 micro L of volkensin (a retrograde suicide transport lectin). Upon challenge with cocaine (15 mg/kg) 12 days after surgery, neither ibotenic acid- nor volkensin-lesioned rats showed any difference in their locomotor response compared with sham controls. These data indicate that bilateral shell lesions do not affect the long-term expression of behavioural sensitization in cocaine-sensitized rats.

Animals↗

A new model for periostracum and shell formation in Unionidae (Bivalvia, Mollusca).

The periostracum in Unionidae consists of two layers. The outer one is secreted within the periostracal groove, while the inner layer is secreted by the epithelium of the outer mantle fold. The periostracum reaches its maximum thickness at the shell edge, where it reflects onto the shell surface. Biomineralization begins within the inner periostracum as fibrous spheruliths, which grow towards the shell interior, coalesce and compete mutually, originating the aragonitic outer prismatic shell layer. Prisms are fibrous polycrystalline aggregates. Internal growth lines indicate that their growth front is limited by the mantle surface. Transition to nacre is gradual. The first nacreous tablets grow by epitaxy onto the distal ends of prism fibres. Later growth proceeds onto previously deposited tablets. Our model involves two alternative stages. During active shell secretion, the mantle edge extends to fill the extrapallial space and the periostracal conveyor belt switches on, with the consequential secretion of periostracum and shell. During periods of inactivity, only the outer periostracum is secreted; this forms folds at the exit of the periostracal groove, leaving high-rank growth lines. Layers of inner periostracum are added occasionally to the shell interior during prolonged periods of inactivity in which the mantle is retracted.

Animals↗

Intravenous cocaine, morphine, and amphetamine preferentially increase extracellular dopamine in the "shell" as compared with the "core" of the rat nucleus accumbens.

The nucleus accumbens is considered a critical target of the action of drugs of abuse. In this nucleus a "shell" and a "core" have been distinguished on the basis of anatomical and histochemical criteria. The present study investigated the effect in freely moving rats of intravenous cocaine, amphetamine, and morphine on extracellular dopamine concentrations in the nucleus accumbens shell and core by means of microdialysis with vertically implanted concentric probes. Doses selected were in the range of those known to sustain drug self-administration in rats. Morphine, at 0.2 and 0.4 mg/kg, and cocaine, at 0.5 mg/kg, increased extracellular dopamine selectivity in the shell. Higher doses of cocaine (1.0 mg/kg) and the lowest dose of amphetamine tested (0.125 mg/kg) increased extracellular dopamine both in the shell and in the core, but the effect was significantly more pronounced in the shell compared with the core. Only the highest dose of amphetamine (0.250 mg/kg) increased extracellular dopamine in the shell and in the core to a similar extent. The present results provide in vivo neurochemical evidence for a functional compartmentation within the nucleus accumbens and for a preferential effect of psychostimulants and morphine in the shell of the nucleus accumbens at doses known to sustain intravenous drug self-administration.

Amphetamine↗

Scaffolding proteins and the genetic control of virus shell assembly.

Historically a gap has existed between the study of the one-dimensional organization of hereditary information in genes, and of the three-dimensional organization of macromolecules in biological structures. In this article we describe progress in closing this gap through the genetic and biochemical analysis of the assembly of the icosahedral shells of spherical viruses, a class of subcellular structures whose subunit organization is relatively well understood. The genes specifying the proteins required for capsid assembly have been identified for many bacterial viruses. By using mutants defective in these genes, it has been possible to identify intermediates in shell morphogenesis and DNA condensation, and to unravel the different levels of the genetic control of macromolecular assembly processes. In general, a precursor shell or procapsid is first constructed, and the DNA is subsequently coiled within it. The construction of a closed shell poses as difficult a problem for a virus as for an architect. In the well-studied bacteriophage P22 of Salmonella typhimurium, the construction of the procapsid requires the interaction of about 200 molecules of the gene-8 scaffolding protein with 420 molecules of the gene-5 coat protein, forming a double-shelled structure with the scaffolding protein on the inside. Once completed, procapsids undergo substantial alteration in the course of encapsulating the viral DNA. In P22, the initiation of DNA packaging triggers the exit of all of the scaffolding molecules from within the capsid, probably through the coat-protein lattice. These released molecules are re-utilized, interacting with newly synthesized coat subunits to form further procapsids. Thus, the scaffolding protein functions catalytically in capsid assembly. All of the well-studied DNA phages require a scaffolding protein species for procapsid assembly, though their properties vary. Purified coat and scaffolding subunits by themselves show little tendency to polymerize, and are stable as monomers in solution. Upon mixing together under the appropriate conditions, however, the proteins copolymerize into double shells. Their interaction with each other appears to be critical for efficient assembly; this interaction probably occurs on the edges of growing shells, and not among subunits in solution. We have termed this kind of process, which we previously described in T4 tail morphogenesis, self-regulated assembly. The subunits are synthesized in a nonreactive form and are activated, not in solution, but upon incorporation into the growing substrate structure. A number of further transformations of the capsid subunits occur only within the organized structure and not as free subunits. Thus, aspects of the genetic information controlling the assembly process are not fully expressed at the level of the properties of protein subunits, but become manifest only through interactions with other proteins, or at a higher level, after completion of the correct organized structure.

Capsid↗

Egg shell quality and cholecalciferol metabolism in aged laying hens.

Calcium-binding protein D28K (calbindin) synthesis, vitamin D metabolism and shell quality were investigated in young and aged laying hens fed diets containing either cholecalciferol (CC) or its 1-hydroxylated derivatives. Duodenal calbindin concentration was similar in the young and in the aged laying hens. Exogenous 1-hydroxylated CC derivatives increased duodenal calbindin concentration, regardless of age. Shell weight and shell density (mg/cm2) were significantly lower (P less than 0.01) in the aged than in the young hens. Egg shell weight and density tended to decrease along the clutch. The rate of decline was higher in aged than in young hens. Feeding aged hens a diet containing 5 micrograms 1,25-dihydroxycholecalciferol [1,25(OH)2CC] or 1 alpha-hydroxycholecalciferol per kilogram improved shell quality, slowed down the progressive reduction in shell quality during the clutch and increased culling and mortality. The results indicate a) that the capacity for expression of 1,25(OH)2D3 in the intestine is not altered by age and b) that prolonged feeding of 1-hydroxylated derivatives of vitamin D3 improves shell quality in aged laying hens and increases culling and mortality.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

Modulation of memory consolidation by the basolateral amygdala or nucleus accumbens shell requires concurrent dopamine receptor activation in both brain regions.

Previous findings indicate that the basolateral amygdala (BLA) and the nucleus accumbens (NAc) interact in influencing memory consolidation. The current study investigated whether this interaction requires concurrent dopamine (DA) receptor activation in both brain regions. Unilateral, right-side cannulae were implanted into the BLA and the ipsilateral NAc shell or core in male Sprague-Dawley rats ( approximately 300 g). One week later, the rats were trained on an inhibitory avoidance (IA) task and, 48 h later, they were tested for retention. Drugs were infused into the BLA and NAc shell or core immediately after training. Post-training intra-BLA infusions of DA enhanced retention, as assessed by latencies to enter the shock compartment on the retention test. Infusions of the general DA receptor antagonist cis-Flupenthixol (Flu) into the NAc shell (but not the core) blocked the memory enhancement induced by the BLA infusions of DA. In the reverse experiment, post-training intra-NAc shell infusions of DA enhanced retention and Flu infusions into the BLA blocked the enhancement. These findings indicate that BLA modulation of memory consolidation requires concurrent DA receptor activation in the NAc shell but not the core. Similarly, NAc shell modulation of memory consolidation requires concurrent DA receptor activation in the BLA. Together with previous findings, these results suggest that the dopaminergic innervation of the BLA and NAc shell is critically involved in the modulation of memory consolidation.

Amygdala↗

Buckling transition in icosahedral shells subjected to volume conservation constraint and pressure: relations to virus maturation.

Minimal energy shapes of closed, elastic shells with 12 pentagonal disclinations introduced in otherwise hexagonally coordinated crystalline lattice are studied. The geometry and the total energy of shells are studied as a function of the elastic properties of the material they are made of. Particular emphasis is put on the buckling transition of the shells, that is, a strong preference of the shell shapes to "buckle out" in spatial regions close to the pentagonal disclinations for a certain range of the elastic parameters of the problem. The transition effectively increases the mean square aspherity of shapes, making them look more like an icosahedron rather than a sphere, which is a preferred shape prior to the onset of the transition. The properties of the buckling transition are studied in cases when (i) the total volume enclosed by the elastic shell has to be fixed and when (ii) there is an internal pressure acting on the shell. This may be related to the maturation process in nonenveloped dsDNA viruses, where the insertion of the genetic material in a preformed protein shell (viral coating) may effectively impose the fixed volume and/or pressure constraint. Several scenarios that may explain the experimentally observed feature of mature viruses being more aspherical (facetted) from their immature precursors are discussed, and predictions for the elastic properties of viral coatings are obtained on the basis of the presented studies.

Computer Simulation↗

On the acoustic diffraction by the edges of benthic shells.

Recent laboratory measurements of acoustic backscattering by individual benthic shells have isolated the edge-diffracted echo from echoes due to the surface of the main body of the shell. The data indicate that the echo near broadside incidence is generally the strongest for all orientations and is due principally to the surface of the main body. At angles well away from broadside, the echo levels are lower and are due primarily to the diffraction from the edge of the shell. The decrease in echo levels from broadside incidence to well off broadside is shown to be reasonably consistent with the decrease in acoustic backscattering from normal incidence to well off normal incidence by a shell-covered seafloor. The results suggest the importance of the edge of the shell in off-normal-incidence backscattering by a shell-covered seafloor. Furthermore, when considering bistatic diffraction by edges, there are implications that the edge of the shell (lying on the seafloor) can cause significant scattering in many directions, including at subcritical angles.

Acoustics↗

Signal processing of the echo signatures returned by submerged shells insonified by dolphin "clicks:" active classification.

A large set of dolphin-emitted acoustic pulses ("echolocation clicks") have been examined, which were reflected from various elastic shells that were suspended, underwater, 4.5 m in front of the animal in a large test site in Kaneohe Bay, Hawaii. A carefully instrumented analog-to-digital system continuously captured the emitted clicks and also the returned, backscattered echoes (A/D conversion at 500 kHz). Using standard conditioning techniques and food reinforces, the dolphin is taught to push an underwater paddle when the "correct" target-the one he has been trained to identify-is presented to him. He communicates his consistently correct identifying choices in this manner. Many echoes returned by three types of cylindrical shells in both the time and frequency domains as well as in the joint time-frequency (t-f) domain, by means of Wigner-type distributions have been examined. It will be shown exactly how specific features observable in these displays are directly related to the physical characteristics of the shells. This processing takes advantage of certain fundamental resonance principles to show which echo features contain information about the size, shape, wall thickness, and material composition of both the shell and its filler substance. In the same fashion that these resonance features give the identifying characteristics of each shell, it is believed they may also give them to the dolphin. These echo features may allow him to extract the target properties by inspection without any need for computations. It is claimed that this may be the fundamental physical explanation of the dolphin's amazing target ID feats, upon which they base their recognition choices. This claim may be substantiated by the detailed analysis of many typical echoes returned by various shells, when they are interrogated by several dolphins. Thus far, this analysis of many echoes from many shells has only been carried out for a single dolphin.

Animals↗

On acoustic scattering by a shell-covered seafloor.

Acoustic scattering by the seafloor is sometimes influenced, if not dominated, by the presence of discrete volumetric objects such as shells. A series of measurements of target strength of a type of benthic shelled animal and associated scattering modeling have recently been completed (Stanton et al., "Acoustic scattering by benthic and planktonic shelled animals," J. Acoust. Soc. Am., this issue). The results of that study are used herein to estimate the scattering by the seafloor with a covering of shells at high acoustic frequencies. A simple formulation is derived that expresses the area scattering strength of the seafloor in terms of the average reduced target strength or material properties of the discrete scatterers and their packing factor (where the reduced target strength is the target strength normalized by the geometric cross section of the scatterers and the averaging is done over orientation and/or a narrow range of size or frequency). The formula shows that, to first order, the backscattering at high acoustic frequencies by a layer of shells (or other discrete bodies such as rocks) depends principally upon material properties of the objects and packing factor and is independent of size and acoustic frequency. Estimates of area scattering strength using this formula and measured values of the target strength of shelled bodies from Stanton et al. (this issue) are close to or consistent with observed area scattering strengths due to shell-covered seafloors published in other papers.

Acoustics↗

Differential conditioned place preference responses to endomorphin-1 and endomorphin-2 microinjected into the posterior nucleus accumbens shell and ventral tegmental area in the rat.

An unbiased conditioned place preference (CPP) paradigm was used to evaluate the reward effects of endogenous mu-opioid receptor ligands endomorphin-1 (EM-1) and endomorphin-2 (EM-2) from the mesolimbic posterior nucleus accumbens (Acb) shell and the ventral tegmental area (VTA) in CD rats. EM-1 (1.6-8.1 nmol) microinjected into posterior Acb shell produced CPP, whereas EM-2 (8.7-17.5 nmol) given into the same Acb shell produced conditioned place aversion (CPA). EM-1 (1.6-16.3 nmol) microinjected into the VTA produced CPP, whereas EM-2 (8.7 and 17.5 nmol) given into the same VTA site did not produce any effect, but at a high dose (35 nmol) produced CPP. EM-1 (3.3 nmol) or EM-2 (17.5 nmol) microinjected into the nigrostriatal substantia nigra was not significantly different from vehicle-injected groups. D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) at 94.13 pmol or 3-methoxynaltrexone at 0.64 pmol microinjected into the posterior Acb shell blocked EM-1-induced CPP and EM-2-induced CPA. At a higher dose, CTOP (941.3 pmol) and 3-methoxynaltrexone (6.4 pmol) produced CPA and CPP, respectively. Coadministration with antiserum against dynorphin A(1-17) (Dyn) (10 microg) microinjected into the posterior Acb shell blocked EM-2-induced CPA. However, it did not affect EM-1-induced CPP. It is concluded that EM-1 and EM-2 produce site-dependent CPP and CPA, respectively, by stimulation of different subtypes of mu-opioid-receptors; stimulation of one subtype of mu-opioid-receptor at the posterior Acb shell and VTA by EM-1 induces CPP, whereas stimulation of another subtype of mu-opioid receptor at the posterior Acb shell, but not the VTA, by EM-2 induces the release of Dyn to produce CPA.

Animals↗

Comparison of standard tube and shell vial cell culture techniques for the detection of cytomegalovirus in clinical specimens.

A monoclonal antibody was used to detect an early antigen of cytomegalovirus (CMV) by fluorescence 16 h after inoculation of MRC-5 monolayers in 1-dram (ca. 3.7-ml) shell vials and low-speed centrifugation. Of 770 specimens (urine, blood, lung tissue, sputum) processed in shell vials, 124 (16%) were positive for the virus at 16 h postinfection. CMV was isolated in standard tube cell cultures (average time, 9 days) from only 88 specimens, but there were no instances (with the exception of 2 blood specimens) in which CMV was recovered from tube cultures but not from shell vials. Additional specimens from 18 patients were positive in the shell vial assay but negative in the conventional tube cell culture assay. Other specimens from 14 of the 18 patients yielded CMV in conventional tube cell cultures. Of the 4 patients from whom CMV was not recovered from other specimens by conventional tube cell culturing, all had evidence of recent CMV infections, as indicated by a fourfold or greater rise in antibody titer. The specificity of the shell vial assay for the detection of CMV is supported by assays of other specimens from the same patients yielding the virus or serological evidence indicating recent infections, the known enhancement of CMV detection after centrifugation of the shell vials, and the distinct and easily recognizable fluorescence confined to the nuclei of CMV-infected cells. Our data indicate that the shell vial cell culture assay for the detection of CMV is as specific as and more sensitive than conventional tube cell culturing for the diagnosis of CMV infections.

Antibodies, Monoclonal↗

Evaluation of number of shell vial cell cultures per clinical specimen for rapid diagnosis of cytomegalovirus infection.

Specimens submitted for the diagnosis of cytomegalovirus (CMV) infection were inoculated into three (blood) or two (urine, tissue, bronchoalveolar lavage [BAL]) shell vials seeded with MRC-5 cells for the diagnosis of CMV infection. We evaluated the detection of 993 specimens that were positive for CMV according to the number of shell vial cell cultures inoculated per specimen. For blood cultures, and considering one CMV-positive shell vial as 100%, inoculation of three shell vials versus one increased the detection rate of the virus by 51%. Inoculation of three shell vials compared with two yielded a 20% increase in the detection rate of CMV. For urine, tissue, and BAL specimens, inoculation of two shell vials compared with one resulted in increases of 7, 10, and 5%, respectively. For maximum detection of CMV in shell vial cell cultures, at least three vials should be inoculated with blood specimens, and two vials should be used for urine, tissue, and BAL samples.

Cell Line↗

Effect of treatment of shell vial cell cultures with dimethyl sulfoxide and dexamethasone for detection of cytomegalovirus.

Urine specimens submitted for the diagnosis of cytomegalovirus infection were inoculated into shell vials that had been pretreated with a combination of dimethyl sulfoxide (DMSO) and dexamethasone (DEX). The results were compared with those for inoculated shell vials which had received no drug treatment. Of 664 specimens, 100 (15%) were positive for cytomegalovirus. Of the 100 strains of cytomegalovirus, 88 (88%) were detected in both DMSO-DEX-treated and untreated shell vials. Of the remaining 12 positive specimens, 6 were detected with untreated shell vials exclusively and 6 were detected with DMSO-DEX-treated shell vials alone (not significant by the sign test). The median number of fluorescent foci was not significantly higher in DMSO-DEX-treated shell vials compared with that in untreated cultures (Wilcoxon signed-rank test; P = 0.1). DMSO-DEX-treated monolayers did not enhance the sensitivity detection of cytomegalovirus in shell vial cell cultures.

Cells, Cultured↗

Randomised controlled trial of breast shells and Hoffman's exercises for inverted and non-protractile nipples.

OBJECTIVE: To determine the value of recommending breast shells or Hoffman's exercises, or both, to pregnant women with inverted or non-protractile nipples who intend to breast feed. DESIGN: Randomised controlled trial with a two treatment by two level factorial design. SETTING: Antenatal clinics in a district general hospital and the community. SUBJECTS: 96 nulliparous women recruited between 25 and 35 completed weeks in a singleton pregnancy with at least one inverted or non-protractile nipple. MAIN OUTCOME MEASURES: Anatomical change of nipples, judged blindly before first breast feeding, and success of breast feeding reported by postal questionnaire six weeks postnatally. RESULTS: Sustained improvement in nipple anatomy was more common in the untreated groups but the differences were not significant (52% (25/48) shells v 60% (29/48) no shells; difference -8% (95% confidence interval -28% to 11%) and 54% (26/48) exercises v 58% (28/48) no exercises; -4% (-24% to 16%)). 24 (50%) women not recommended shells and 14 (29%) recommended shells (21%; 40% to 2%) were breast feeding six weeks after delivery (p = 0.05), reflecting more women recommended shells both deciding to bottle feed before delivery and discontinuing breast feeding. The same number of women in exercise and no exercise groups were successfully breast feeding (0%; -20% to 20%). 13% of women approached about the trial (and planning to breast feed) did not attempt breast feeding. CONCLUSIONS: Recommending nipple preparation with breast shells may reduce the chances of successful breast feeding. While there is no clear evidence that the treatments offered are effective antenatal nipple examination should be abandoned.

Adolescent↗