Cellular Uptake and Efflux of Palbociclib In Vitro in Single Cell and Spheroid Models.

Adequate drug distribution through tumors is essential for treatment to be effective. Palbociclib is a cyclin-dependent kinase 4/6 inhibitor approved for use in patients with hormone receptor positive, human epidermal growth factor receptor 2 negative metastatic breast cancer. It has unusual physicochemical properties, which may significantly influence its distribution in tumor tissue. We studied the penetration and distribution of palbociclib in vitro, including the use of multicellular three-dimensional models and mathematical modeling. MCF-7 and DLD-1 cell lines were grown as single cell suspensions (SCS) and spheroids; palbociclib uptake and efflux were studied using liquid chromatography-tandem mass spectrometry. Intracellular concentrations of palbociclib for MCF-7 SCS (C max 3.22 µM) and spheroids (C max 2.91 µM) were 32- and 29-fold higher and in DLD-1, 13- and 7-fold higher, respectively, than the media concentration (0.1 µM). Total palbociclib uptake was lower in DLD-1 cells than MCF-7 cells in both SCS and spheroids. Both uptake and efflux of palbociclib were slower in spheroids than SCS. These data were used to develop a mathematical model of palbociclib transport that quantifies key parameters determining drug penetration and distribution. The model reproduced qualitatively most features of the experimental data and distinguished between SCS and spheroids, providing additional support for hypotheses derived from the experimental data. Mathematical modeling has the potential for translating in vitro data into clinically relevant estimates of tumor drug concentrations. SIGNIFICANCE STATEMENT: This study explores palbociclib uptake and efflux in single cell suspension and spheroid models of cancer. Large intracellular concentrations of palbociclib are found after drug exposure. The data from this study may aid understanding of the intratumoural pharmacokinetics of palbociclib, which is useful in understanding how drug distributes within tumor tissue and optimizing drug efficacy. Biomathematical modelling has the potential to derive intratumoural drug concentrations from plasma pharmacokinetics in patients.

Short communication: Reproduction outcomes in dairy heifers following a 14-d progesterone insert presynchronization protocol.

The objectives were to evaluate pregnancy per artificial insemination (AI), days to first AI, and proportion pregnant within 7 d of AI eligibility in dairy heifers subjected to presynchronization compared with dairy heifers not presynchronized. Thirty days before AI eligibility, Holstein heifers were assigned randomly to 1 of 3 groups: 14-d controlled internal drug release (CIDR; containing progesterone) presynchronization, PGF2α presynchronization, or control (no presynchronization). Heifers in the 14-d CIDR presynchronization treatment (n = 119) received a CIDR on d -30, which was removed on d -16, followed by an injection of PGF2α upon entry to the breeding program (d 0). Heifers in the PGF2α presynchronization treatment (n = 118) received an injection of PGF2α on d -11 and d 0. Control heifers (n = 121) were not presynchronized and received an injection of PGF2α on d 0. All heifers received tail paint on d 0 to facilitate once-daily detection of estrus (based on paint removal). Heifers detected in estrus received AI with conventional semen on the same morning as detected estrus. Generalized linear mixed models were used to assess mean treatment differences. Following PGF2α treatment on d 0, more heifers were detected in estrus in the first 7 d after eligibility in the 14-d CIDR group (95.8%) compared with the PGF2α (74.6%) and control (66.9%) groups. Days to first AI differed between treatments (14-d CIDR = 3.6 d vs. PGF2α = 5.0 d vs. control = 6.8 d). Pregnancy per AI for first AI within 7 d of eligibility was 71.9% (14-d CIDR), 58.0% (PGF2α), and 61.7% (control), and differed between 14-d CIDR and PGF2α heifers. Presynchronization with a 14-d CIDR increased the proportion of heifers pregnant in the first 7 d of eligibility (14-d CIDR = 68.9% vs. PGF2α = 43.2% vs. control = 41.3%). Projected days on feed (d 0 to projected calving date) were 295 (14-d CIDR), 302 (PGF2α), and 305 (control), and were different between the 14-d CIDR and control heifers. The potential economic benefit to the producer was $15.85 per heifer presynchronized with a 14-d CIDR protocol compared with the control group. Treatment of dairy heifers with a 14-d CIDR effectively presynchronized estrus, resulting in a greater proportion detected in estrus, reduced days to first AI, and an increased proportion of heifers pregnant within the first 7 d after breeding eligibility compared with heifers presynchronized with a single PGF2α injection and control heifers.

Pregnancy outcomes are not improved by administering gonadotropin-releasing hormone at initiation of a 5-day CIDR-Cosynch resynchronization protocol for lactating dairy cows.

Using a 5-d controlled internal drug-release (CIDR)-Cosynch resynchronization protocol, the objective of this study was to determine the effect of the initial GnRH injection on pregnancy per artificial insemination (P/AI) to the second artificial insemination in lactating Holstein dairy cows. On 37 ± 3 d (mean ± standard deviation) after the first artificial insemination, and upon nonpregnancy diagnosis (d 0 of the experiment), lactating cows eligible for a second artificial insemination (n = 429) were enrolled in a 5-d CIDR-Cosynch protocol. On d 0, all cows received a CIDR insert and were assigned randomly to receive the initial GnRH injection (GnRH; n = 226) of the protocol or no-GnRH (n = 203). Blood samples were collected from a sub-group of cows (n = 184) on d 0 and analyzed for progesterone (P4) concentration. On d 5, CIDR inserts were removed, and all cows received 1 injection of PGF2α. On d 6 and 7, cows were observed once daily by employees for tail-chalk removal, and cows detected in estrus on d 6 or 7 received artificial insemination that day (EDAI), and did not receive the final GnRH injection. The remaining cows not detected in estrus by d 8 received GnRH and timed artificial insemination (TAI). Pregnancy status was confirmed by transrectal palpation of uterine contents at 37 ± 3 d (mean ± standard deviation) after the second artificial insemination. Eliminating the initial GnRH injection had no effect on P/AI compared with cows receiving GnRH (27 vs. 21%), respectively. Similarly, method of insemination (EDAI vs. TAI) and its interaction with treatment had no effect on P/AI. Primiparous cows had greater P/AI than multiparous cows (31 vs. 21%). Mean P4 concentrations (n = 184) at the initiation of the protocol did not differ between treatments (4.51 ± 0.35 ng/mL no-GnRH vs. 3.96 ± 0.34 ng/mL of GnRH). When P4 concentrations were categorized as high (≥1 ng/mL) or low (<1 ng/mL), P/AI tended to be greater for high P4 concentrations (n = 136) compared with low (n = 48) P4 concentrations (26 vs. 16%, respectively). No differences were observed in the proportion of cows with high or low P4 between treatments. Collectively, these results provide evidence that eliminating the initial GnRH in a 5-d CIDR-Cosynch resynchronization protocol for lactating dairy cows did not reduce P/AI in this study.

Transvaginal ultrasound (TVUS)-guided biopsy is safe and effective in diagnosing peritoneal carcinomatosis and recurrent pelvic malignancy.

AIM: To assess safety and accuracy of transvaginal ultrasound (TVUS)-guided biopsy in achieving a diagnosis of peritoneal carcinomatosis (PC). MATERIALS AND METHODS: This was a retrospective study comprising a cohort of 54 consecutive women aged 18-85 years referred from the gynaecological oncology multidisciplinary team meeting (MDTM) who attended for TVUS-guided biopsy procedures in a tertiary oncology centre over a 4-year period (2010-2014). Clinicopathological validation was assessed using online patient records and radiological information systems. An independent oncologist assessed patient outcomes. RESULTS: The procedure was successful in all 19 patients with suspected recurrent malignancy with diagnosis validated against previous histology. Successful histological confirmation was achieved in 31 of 35 patients with suspected PC, which was thereafter validated by histology from subsequent surgery and favourable response to site-specific therapies (n=22). In three patients with suspected PC, the procedure did not result in biopsy as a suitable target could not be identified. Another woman had two false-negative biopsies. Thus overall a site-specific and subtype cancer diagnosis was obtained for 50 women giving an overall patient success rate of 93% (50/54). There were no procedure-related complications. CONCLUSION: TVUS core biopsy is a safe, effective, well-tolerated, and valuable technique in modern oncological management of PC when other diagnostic options are unavailable.

Pregnancy per artificial insemination during summer in lactating dairy cows after treatment with aspirin.

Embryonic loss in cattle may be related to a hormonal imbalance resulting in alterations in timing of prostaglandin F2α (PGF2α) secretion around the time of maternal recognition of pregnancy. The objective of this study was to examine effects of aspirin (a PGF2α inhibitor) on pregnancy per AI (P/AI), and progesterone (P4), and pregnancy specific protein B (PSPB) concentrations in lactating dairy cows inseminated more than once after parturition. Fourteen days after second or subsequent AI (Day 0 = Day of AI), 556 cows were assigned randomly to aspirin (187.2 g total; n = 277) or control (n = 279) groups. Aspirin was administered orally on Day 14 and 15, and control cows were subjected to sham bolus administration. On Day 25, blood samples were collected from a subset of cows (n = 194) to quantify P4 and PSPB, whereas pregnancy was determined in all cows at 35-42 days post-AI. Maximum daily ambient temperature ranged from 38-41 °C during the experiment. Mean parity, days in milk, and times bred before treatment (TBRD) did not differ between groups. There were no differences in P/AI between treatments (aspirin 21.6 % compared with control 27.5 %). Neither treatment, parity, TBRD, or any two-way interactions with treatment affected concentrations of P4. Moreover, there were no effects (P > 0.50) of treatment, or treatment by TBRD interaction on serum PSPB concentrations. A tendency (P = 0.07) occurred for multiparous cows to have greater serum PSPB concentrations compared with primiparous cows. Mean serum PSPB concentrations tended (P = 0.07) to be greater for second or third TBRD compared to fourth and greater TBRD. These results provide evidence that aspirin administered during periods of heat stress after the second and subsequent AI post-partum during the summer months does not improve P/AI or alter P4 and PSPB in lactating dairy cows.

Regulation of microtubule dynamics and myogenic differentiation by MURF, a striated muscle RING-finger protein.

The RING-finger domain is a novel zinc-binding Cys-His protein motif found in a growing number of proteins involved in signal transduction, ubiquitination, gene transcription, differentiation, and morphogenesis. We describe a novel muscle-specific RING-finger protein (MURF) expressed specifically in cardiac and skeletal muscle cells throughout pre- and postnatal mouse development. MURF belongs to the RING-B-box-coiled-coil subclass of RING-finger proteins, characterized by an NH(2)-terminal RING-finger followed by a zinc-finger domain (B-box) and a leucine-rich coiled-coil domain. Expression of MURF is required for skeletal myoblast differentiation and myotube fusion. The leucine-rich coiled-coil domain of MURF mediates association with microtubules, whereas the RING-finger domain is required for microtubule stabilization and an additional region is required for homo-oligomerization. Expression of MURF establishes a cellular microtubule network that is resistant to microtubule depolymerization induced by alkaloids, cold and calcium. These results identify MURF as a myogenic regulator of the microtubule network of striated muscle cells and reveal a link between microtubule organization and myogenesis.

Staging of endometrial cancer with MRI: guidelines of the European Society of Urogenital Imaging.

The purpose of this study was to define guidelines for endometrial cancer staging with MRI. The technique included critical review and expert consensus of MRI protocols by the female imaging subcommittee of the European Society of Urogenital Radiology, from ten European institutions, and published literature between 1999 and 2008. The results indicated that high field MRI should include at least two T2-weighted sequences in sagittal, axial oblique or coronal oblique orientation (short and long axis of the uterine body) of the pelvic content. High-resolution post-contrast images acquired at 2 min +/- 30 s after intravenous contrast injection are suggested to be optimal for the diagnosis of myometrial invasion. If cervical invasion is suspected, additional slice orientation perpendicular to the axis of the endocervical channel is recommended. Due to the limited sensitivity of MRI to detect lymph node metastasis without lymph node-specific contrast agents, retroperitoneal lymph node screening with pre-contrast sequences up to the level of the kidneys is optional. The likelihood of lymph node invasion and the need for staging lymphadenectomy are also indicated by high-grade histology at endometrial tissue sampling and by deep myometrial or cervical invasion detected by MRI. In conclusion, expert consensus and literature review lead to an optimized MRI protocol to stage endometrial cancer.

Discriminative MRI features of fallopian tube masses.

Fallopian tube disease, both acute and chronic, is a common cause of a sonographically indeterminate adnexal mass and may mimic ovarian cancer. Magnetic resonance imaging (MRI) is now widely used as a problem-solving tool in these circumstances. The purpose of this review is to provide the discriminative MRI features of Fallopian tube masses and illustrate the key signs that establish their origin and nature. Familiarity with these characteristics enables distinction of tubal disease from malignant adnexal disease with major impact on management. On MRI, Fallopian tube disease exhibits features that parallel the classical sonographic findings, but which can be more reliably recognized due to improved contrast and spatial resolution, multiplanar capacity, effective field of view, and tissue characterization. Recognition of these characteristic morphological features and specific MRI signal patterns are key to a specific diagnosis. The anatomical and histopathological basis of these MRI signs is emphasized, covering also the differential diagnosis and pitfalls. Two new signs, "synechiae" and "amorphous shading", are also described that have not been well described previously in MRI of tubal disease.

Indeterminate lesions in cancer imaging.

Indeterminate lesions are detected on cancer imaging examinations at all points along the patient pathway. Decisions made about indeterminate lesions may have profound impact on patient management, particularly when these may represent solitary sites of metastasis. In this process of decision making the radiologist, having detected a potential metastasis, must attempt to characterize the abnormality and then to make a judgement about its impact on management based on knowledge of the clinical context. Not every abnormality can be confidently characterized using the modality of detection and the indeterminate lesion may require further clarification through discussion in the format of a multidisciplinary meeting, planned further investigation, or intervention. In some circumstances the lesion remains indeterminate and a monitoring approach is appropriate. Uncertainty must not be regarded as a personal weakness of any individual, but may remain even after collective discussion in a multidisciplinary setting. The aim must be to develop a plan of action (or inaction) that is understandable, useful, and acceptable to the patient and clinician. In this review, the principles guiding these decisions are discussed in further detail and practical solutions to some commonly encountered indeterminate lesions are suggested, focusing on those that might represent solitary sites of disease and whose management impact may be profound.

Basic fibroblast growth factor activates serum response factor gene expression by multiple distinct signaling mechanisms.

Serum response factor (SRF) plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. It is a key regulator of many cellular early response genes which are believed to be involved in cell growth and differentiation. The mechanism by which SRF activates transcription in response to mitogenic agents has been extensively studied; however, significantly less is known about regulation of the SRF gene itself. Previously, we identified distinct regulatory elements in the SRF promoter that play a role in activation, including a consensus ETS domain binding site, a consensus overlapping Sp/Egr-1 binding site, and two SRF binding sites. We further showed that serum induces SRF by a mechanism that requires an intact SRF binding site, also termed a CArG box. In the present study we demonstrate that in response to stimulation of cells by a purified growth factor, basic fibroblast growth factor (bFGF), the SRF promoter is upregulated by a complex pathway that involves at least two independent mechanisms: a CArG box-independent mechanism that is mediated by an ETS binding site, and a novel CArG box-dependent mechanism that requires both an Sp factor binding site and the CArG motifs for maximal stimulation. Our analysis indicates that the CArG/Sp element activation mechanism is mediated by distinct signaling pathways. The CArG box-dependent component is targeted by a Rho-mediated pathway, and the Sp binding site-dependent component is targeted by a Ras-mediated pathway. Both SRF and bFGF have been implicated in playing an important role in mediating cardiogenesis during development. The implications of our findings for SRF expression during development are discussed.

Expression of the SRF gene occurs through a Ras/Sp/SRF-mediated-mechanism in response to serum growth signals.

Serum Response Factor (SRF) plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. It is a key regulator of many cellular early response genes which are believed to be involved in cell growth, differentiation, and development. The mechanism by which SRF activates transcription in response to mitogenic agents has been extensively studied, however, less is known about regulation of the SRF gene itself. Previously, we identified distinct regulatory elements in the SRF promoter that play a role in activation, including an ETS domain binding site, an overlapping Sp1/Egr-1 binding site, and two SRF binding sites. We further showed that serum induces the SRF gene by a mechanism that requires an intact SRF binding site, also termed a CArG box. In the present study we demonstrate that in response to stimulation by cells by lysophosphatidic acid (LPA) or whole serum, the SRF promoter is upregulated by a bipartite pathway that requires both an Sp1 factor binding site and the CArG motifs for maximal stimulation. The CArG box-dependent component of this pathway is targeted by Rho mediated signals, and the Sp1 binding site dependent component is targeted by Ras mediated signals.

UB-165: a novel nicotinic agonist with subtype selectivity implicates the alpha4beta2* subtype in the modulation of dopamine release from rat striatal synaptosomes.

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the alpha3beta2-selective alpha-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this alpha-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the alpha-conotoxin-MII-insensitive subtype is composed of alpha4 and beta2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at alpha4beta2* and alpha3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over alpha7-type and muscle binding sites. (+/-)-Epibatidine, (+/-)-anatoxin-a, and (+/-)-UB-165 stimulated [(3)H]-dopamine release from striatal synaptosomes with EC(50) values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. alpha-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (+/-)-UB-165 is a very poor agonist at the alpha-conotoxin-MII-insensitive nAChR subtype. In assays of (86)Rb(+) efflux from thalamic synaptosomes, a model of an alpha4beta2* nAChR response, (+/-)-UB-165 was a very weak partial agonist; the low efficacy of (+/-)-UB-165 at alpha4beta2 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (+/-)-UB-165 and (+/-)-anatoxin-a were similarly efficacious and similarly sensitive to alpha-conotoxin-MII in increasing intracellular Ca(2+) in SH-SY5Y cells, a functional assay for native alpha3-containing nAChR. These data support the involvement of alpha4beta2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.

Stable isotopic composition of the active pharmaceutical ingredient (API) naproxen.

A survey of the multi-stable isotopic composition of an active pharmaceutical ingredient (API), naproxen, was performed to assess the potential of Isotope Ratio Mass Spectrometry (IRMS) to distinguish the provenance of APIs. Twenty-six lots of naproxen from six manufacturers representing four countries (Italy, India, Ireland, and the USA) were analyzed for three isotope ratios (13C/12C, 18O/16O, and D/H). The samples were analyzed by either Elemental Analyzer/Isotope Ratio Mass Spectrometry (EA/IRMS: carbon (delta13C)) or by Thermal Conversion-EA/IRMS (TCEA/IRMS: hydrogen (deltaD) and oxygen (delta18O)). Bivariate and trivariate isotope ratio graphs for naproxen show marked clustering of the data for five out of the six naproxen manufacturers, suggesting that IRMS may be a plausible means to screen for manufacturer of given APIs.

Interleukin-1 activates protein kinase A and stimulates adrenocorticotropic hormone release from AtT-20 cells.

Studies from this and other laboratories have shown that interleukin-1 (IL-1) stimulates ACTH secretion directly from AtT-20 cells. The present studies were conducted to determine the signal transduction mechanisms activated by IL-1 to stimulate ACTH release. IL-1 significantly (P < 0.05) elevated ACTH release after incubation periods of 4, 8, and 24 h. IL-1-induced ACTH release was not additive to that of CRF, cholera toxin, 8-bromo-cAMP, or forskolin. In contrast, IL-1 and the phorbol ester phorbol 12-myristate 13-acetate together produced a greater increase (P < 0.05) in ACTH release than either agent alone. IL-1 did not stimulate cAMP accumulation at any time period between 5 min and 24 h and did not affect cAMP accumulation induced by CRF, cholera toxin, or forskolin. The lack of additivity between IL-1 and CRF, cholera toxin, 8-bromo-cAMP, and forskolin suggests that IL-1 stimulates ACTH release by a pathway that shares some common step(s) with CRF. Because IL-1 did not affect cAMP accumulation, the effect of IL-1 on protein kinase A (PKA) was investigated. IL-1 began to increase (P < 0.05) PKA activity at 15 min and remained elevated for 2 h before returning to control levels. IL-1 stimulation of PKA and the lack of additivity between IL-1 and CRF, forskolin, and cholera toxin indicate that PKA is the intracellular mediator used by IL-1 to stimulate ACTH release in AtT-20 cells.

Tissue-specific expression of insulin-like growth factor binding protein-3 protease activity during rat pregnancy.

The abundances of insulin-like growth factor binding proteins (IGFBPs) in sera and tissue homogenates of rats at days 12, 15, and 18 of pregnancy were determined by ligand- and immunoblotting. As in serum, IGFBP-3 was abundant in day 12 uterus, placenta, and fetuses, and decreased by day 15. On day 18 of pregnancy, IGFBP-2 was predominant and IGFBP-3 was less than 25% of day 12 values in fetus and placenta, and was undetectable in uterus and decidua. In contrast, IGFBPs in the nonreproductive tissues did not change significantly. IGFBP-3 was more abundant in muscle than heart and liver, and was not detected in lung, kidney, or brain. The decrease in IGFBP-3 in serum and reproductive tissues between days 12 and 15 of pregnancy was temporally related to the appearance of IGFBP-3 protease activity. Proteolytic activity was detectable only at low levels in brain, liver, and spleen, and was undetectable in lung, heart, muscle, and kidney. The specific protease inhibitors that blocked IGFBP-3 proteolytic activities in pregnant rat serum and decidua were virtually identical and suggested inhibition of a divalent cation-dependent tryptic-like serine protease. Furthermore, exposure of bovine IGFBP-3 (in nonpregnant bovine serum) or radiolabeled recombinant human IGFBP-3 to day 18 pregnant rat serum, decidua or uterus resulted in the generation of IGFBP-3 fragments with the same apparent Mrs (29-31 K and 18-23 K). We postulate that tissue-specific degradation may be as important as synthesis in determining IGFBP-3 abundance, and that the dramatic changes in IGFBPs in reproductive and fetal tissues may cause changes in IGF availability which are necessary for rapid tissue growth and differentiation.

Decline in respiratory control ratio of rat liver mitochondria in old age.

Rat liver mitochondria isolated from old animals (27-33 months) showed a clear decline in the state 3 rate of respiration (presence of ADP) compared with mitochondria from mature animals (3-12 months) when using either succinate or NAD+-linked substrates. The state 4 rate of respiration (absence of ADP), which is a sensitive indicator of damage to the inner mitochondrial membrane, remained unchanged. Consequently the respiratory control ratio (state 3/state 4) declined. A loss of reserve capacity to maintain the ADP:O ratio was also observed. These observations indicate a decline in energy production in old animals.

Prevalence of antibodies to Neospora sp. in horses from Alabama and characterisation of an isolate recovered from a naturally infected horse [corrected].

An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of the 536 serum samples. Endpoint titres for the positive samples were 1:50 (35/6.5%), 1:100 (19/3.5%), 1:200 (7/1.3%) and 1:1600 (1/0.2%). Tachyzoites were first seen in cultured bovine turbinate cells 32 days after inoculation with spinal cord homogenates from the horse with CNS disease. Tachyzoites reacted with known N. caninum-positive serum from horses, cows, dogs and mice, but did not react with murine anti-Toxoplasma gondii or equine anti-Sarcocystis neurona serum. Ultrastructural features of tachyzoites and results of comparison of tachyzoite immunodominant proteins revealed that they were identical to those of N. hughesi, a species described recently from a naturally infected horse. The isolate recovered from the naturally infected horse in the present study (designated NA1) is thought to be an isolate of N. hughesi, although confirmation of this awaits additional molecular characterisation. These results provide some additional evidence that N. hughesi is a valid species and that Neospora infections in horses may occur in widely separated geographic regions of the United States.

Directed deprotonation-transmetalation as a route to substituted pyridines.

Regioselective C-4 deprotonation of 3-bromopyridine, followed by Li/Zn transmetalation and Pd-mediated coupling processes, provides a flexible entry to 4-substituted and 3,4-disubstituted pyridines. Application of a similar sequence to 2-bromopyridine (with LDA as base) provides 2,3-disubstituted pyridines, but using lithium 2,2,6,6-tetramethylpiperidide (LiTMP) provides access to both the 2,3- and 2,4-disubstituted isomers.

Variability of continuous-wave Doppler flow velocity waveform indices from the umbilical artery.

Using continuous-wave Doppler ultrasound, the reproducibility of umbilical artery blood flow velocity waveforms, recorded by two operators, was evaluated in 20 high-risk antenatal inpatients. Inter-operator variability was not significant for any index. The intra-operator coefficients of variation for resistance index, pulsatility index, and systolic/diastolic ratio were 7 and 7%, 12 and 12%, and 15 and 11%, respectively, for each operator. The intra-class coefficient of reliability, calculated by analysis of variance, indicated that the contribution of inter-patient variability to total variance was four times that of intra-operator variability.

Identification of fetal growth retardation: comparison of Doppler waveform indices and serial ultrasound measurements of abdominal circumference and fetal weight.

OBJECTIVE: To compare the serial ultrasound assessment of abdominal circumference (AC) and fetal weight versus single values of umbilical artery pulsatility index (PI) and the aortic-middle cerebral PI ratio in the identification of fetal growth retardation (FGR). METHODS: Serial AC measurements and estimates of weight were obtained in 104 small fetuses in the third trimester of pregnancy. These serial values (expressed as a change in standard deviation [SD] scores) were compared with the final value of AC and estimated fetal weight (EFW), umbilical artery PI, and the aortic-middle cerebral artery PI ratio (all expressed as SD scores) for their ability to predict a reduced neonatal ponderal index, mid-arm circumference-head circumference (HC) ratio, and skinfold thickness. Receiver-operating characteristic (ROC) curves were derived and the ultrasound measurements evaluated by calculating the areas under the ROC curves. RESULTS: Serial estimates of fetal weight resulted in a significantly larger area under the ROC curve compared with the final aortic-middle cerebral PI ratio in the prediction of an abnormal mid-arm circumference-HC ratio and compared with the final AC, EFW, and umbilical artery PI in the prediction of all neonatal morphometric indices. CONCLUSION: In small fetuses, fetal growth failure as determined by the serial assessment of AC and EFW was superior to the final AC and EFW, umbilical artery PI, and the aortic-middle cerebral PI ratio in the prediction of abnormal neonatal morphometry indicative of FGR.