Batrachochytrium dendrobatidis. Understanding, predicting, and managing the impacts of chytridiomycosis on any amphibian species will require detailed information on its ecology and behavior because this pathogen is transmitted by contact with water or other individuals, and pathogen growth rates are thermally sensitive. The common mistfrog (Litoria rheocola) is an endangered tropical rainforest frog that has declined due to chytridiomycosis. We tracked L. rheocola during the winter (cool/dry) and summer (warm/wet) seasons at a low- and high-elevation site. We found that seasonal differences in environmental temperatures and frog behavior should render this species most vulnerable to B. dendrobatidis during cooler months and at higher elevations, which matches observed patterns of infection prevalence in this species. During winter, frogs moved shorter distances than during summer, and they spent less time in vegetation and more time in the stream, which should increase exposure to aquatic B. dendrobatidis zoospores. At a low-elevation site (40 m ASL), estimated body temperatures were within the optimal range for B. dendrobatidis growth (15-25°C) most of the time during winter, but they reached temperatures above this threshold frequently in summer. At a higher elevation (750 m ASL), estimated body temperatures were within the range most favorable for B. dendrobatidis year-round, and did not exceed 25°C, even during summer. Our study provides the first detailed information on the ecology and behavior of L. rheocola and suggests ecological mechanisms for infection dynamics that have been observed in this endangered species.One of the most devastating wildlife diseases ever recorded is chytridiomycosis, a recently emerged amphibian disease that is caused by the chytrid fungus  Batrachochytrium dendrobatidis; it has caused severe amphibian declines and extinctions in many regions of the world [Amphibians have experienced rapid population declines and species extinctions in recent decades, and one-third of extant amphibians are classified as threatened . Althoughe world ,5. This he world .B. dendrobatidis year-round, but they are often most vulnerable during cooler months and at higher elevations, particularly in tropical regions [B. dendrobatidis on environmental conditions; this pathogen is highly sensitive to desiccation [B. dendrobatidis by affecting rates of transmission and the buildup of infections on their skin. Even closely related frog species that occur at the same sites can have very different patterns of movement, habitat use, and social behavior, all of which can influence susceptibility to B. dendrobatidis [B. dendrobatidis [B. dendrobatidis growth and survival, are more likely to develop and maintain infections [In many regions, amphibians are infected by  regions –9. Theseiccation  and its iccation ,12). Beciccation –9,13. Thobatidis ,15. Becaobatidis , speciesobatidis ,17. Addifections ,19.Litoria rheocola), an IUCN Endangered species [Litoria rheocola is a small treefrog . The Queensland Department of Environment and Resource Management also approved all animal protocols and provided permission to work at all field locations (permit WITK03070508).We conducted our study at two rainforest streams that differed substantially in elevation; Frenchman Creek  and Windin Creek  are both located in Wooroonooran National Park, Queensland, Australia. Both streams are surrounded by tropical rainforest, characterized by dense vegetation, including large trees (10 m in height), vines, epiphytes, shrubs, and herbaceous plants. The streams vary in width (5–20 m), contain pools, runs, riffles, and waterfalls, and the streambeds consist of rocks that range in size from small pebbles to large boulders (10 m in diameter). At each site, we captured and tracked frogs along a 400-m section of stream over a three-week period during the winter (cool/dry season) in 2009 , and the summer (warm/wet season) in 2010 .B. dendrobatidis because infection by this pathogen can alter amphibian behavior [B. dendrobatidis at first capture, and a second sample was taken from recaptured frogs at the end of the study period to determine whether they had become infected. We swabbed the ventral surface and all four feet of each frog with a sterile rayon swab, covering these areas twice. Swab samples were analyzed using real-time quantitative PCR assays [For the present study, we examined data only on tracked frogs that were not infected by behavior ,34–36, aR assays . Frogs tWe tracked a total of 76 uninfected frogs: 42 during winter  and 34 during summer . Following capture, we recorded each frog’s sex , body mass, and snout-urostyle length. We included both males and females in all analyses because females only represented a small proportion of the individuals that we tracked , and were not overrepresented in any tracking period (maximum of N = 3 per tracking period); therefore, any gender effects should not be confounded with treatment effects.L. rheocola are too small to carry radiotransmitters, we tracked frogs using harmonic direction finding [After each frog was processed following capture, it was immediately fitted with an external tracking device. Because  finding ,33. Trac finding . We buil finding . The tub finding . We excl finding ,33.L. rheocola has strong site fidelity and when a frog was not found on a particular survey (or surveys), it was almost always subsequently found less than 2 m from its most recent known location. This suggests that frogs were moving short distances and sheltering beneath rocks when we could not detect them. Because we were not able to locate all frogs on all surveys, sample sizes vary among analyses, based on available data. We attempted to meet the assumptions of all statistical analyses, but we used raw data in cases when transformations did not result in normal distributions because ANOVA is robust to deviations from normality [We tracked frogs using RECCO detectors . These hand-held devices act as both transmitters and receivers; they emit a continuous signal that is absorbed and re-emitted at a higher frequency by the diodes. We attempted to locate frogs once during each day (10:00–17:00) and once each night (20:00–03:00) throughout the tracking period. To do this, we walked slowly along the center and edges of the stream and used the detector to scan all areas potentially used by frogs, including rocks in the stream and vegetation along the stream edge. At the end of the tracking period, we removed the tracking devices from all recaptured frogs (N = 19); the harnesses were designed to fall off the remaining frogs after approximately 21 days as the fine cotton thread deteriorated. Using harmonic direction finding to locate animals is not as effective as radiotelemetry; the tracking detector typically has a maximum detection range of 15 m at rainforest streams and cannot penetrate rock . Howeverormality .Litoria rheocola is a treefrog, and individuals move along and at right angles to the stream and also climb up and down vegetation; therefore, they use all three dimensions of space, with their directions of movement largely unconstrained in the horizontal plane but largely restricted to movements up and down individual plants in the vertical direction. Because of these movement patterns, we recorded horizontal and vertical displacement from the previous location separately (to the nearest 5 cm). Movement distances were calculated only when individuals were located on consecutive surveys ; when frogs were not located on consecutive surveys, movement distances for the time interval concerned were recorded as missing values and were not included in any analyses. We also determined the probability of movement from day or night locations by calculating the movement probability for each frog . We used a two-way MANOVA to analyze the distances moved by individual frogs between day and night locations, and the probability of moving from these locations. We used season (winter or summer) and site (Frenchman Creek or Windin Creek) as independent variables, and the median horizontal distance between locations, median vertical distance between locations, and movement probability of each frog as dependent variables.We examined frog movements at two scales: daily displacement of frogs, and total displacement of frogs over the three-week study period. Each time we located a frog, we marked its location using flagging tape, and recorded its distance along the transect. We also measured the frog's height above the stream, and its horizontal distance from the stream edge. To investigate the daily movements of frogs, we measured the distances moved by frogs between consecutive locations; this includes movement from each nocturnal perch site to the subsequent diurnal shelter site, and from each diurnal shelter site to the subsequent nocturnal perch site. We examined longer-term movements of frogs by determining their displacement along the stream during our three-week study period. For each frog, we calculated the difference between the minimum and maximum distances along our stream transect at which the frog was observed. We analyzed these data using a two-way ANOVA with season and site as independent variables, and total displacement of each frog as the dependent variable.We also studied the positions of frogs in relation to the stream to determine whether their proximity to the stream varied by season and site. We used two-way MANOVAs to analyze these data. Season and site were independent variables, and median height above stream, and median horizontal distance from the stream edge of each frog were dependent variables. Separate analyses were performed for day and night locations.L. rheocola body temperatures [We used physical models that mimic the thermal and hydric properties of frogs ,43 to esB. dendrobatidis in culture: <15°C, 15–25°C, and >25°C [B. dendrobatidis in northeastern Queensland is fastest between 15°C and 25°C in culture, and is slower outside of this temperature range [B. dendrobatidis in culture and on amphibian hosts [We used all thermal data collected for each frog to calculate the proportion of its body temperatures that occurred in temperature categories that are relevant to the growth of nd >25°C ,12. GrowEach time we located a frog, we recorded the substrate that the ventral surface of the frog was contacting. We defined four substrate categories: vegetation, rock, leaf litter, and soil/coarse woody debris. We also recorded whether the substrate was dry or wet (from the stream or rain), and whether the frog was in a sheltered or exposed position. For each frog, we calculated the proportion of locations in each substrate category, the proportion of locations that were wet, and the proportion that were sheltered. We performed separate calculations for day and night locations. To analyze microhabitat data, we used multi-response permutation procedures (MRPP) and Monte Carlo re-sampling with 10,000 iterations . We perWe also estimated the rate of evaporative water loss in each microhabitat used by each frog by weighing the permeable physical model (described above) to the nearest 0.1 g immediately before and after placement in each frog location, and calculating the proportion of mass lost over 48 hr ,46. For We studied nocturnal perch-site selection by frogs during winter and summer. Because of obvious seasonal differences in selected perch sites, we used different approaches for each season. During winter, we measured the following characteristics of vegetation used by frogs at each perch site: distance from the stream edge, distance to the nearest riffle, plant height, area covered by the plant’s canopy (average length × width), stream depth, and number of rocks within 1 m. We also measured these same characteristics for vegetation that was available to frogs, but was not used by frogs during our study; we selected the nearest plant to the stream every 10 m along both sides of a 200-m section of stream for measurement. To determine whether frogs selected perch sites that were different from available perch sites, we analyzed our data using a two-way MANOVA. We used perch type (used or available) and site (Frenchman Creek or Windin Creek) as independent variables, and the six characteristics described above as dependent variables.During summer, frogs were often observed in taller vegetation than during winter, especially in taller trees. To determine whether this seasonal difference was statistically significant, we compared the height of vegetation that frogs perched on during the two seasons. We used a two-way ANOVA with season and site as independent variables, and vegetation height as the dependent variable. To understand whether frogs selected trees that were significantly different from available trees during summer, we measured the following characteristics of all trees used by frogs: tree height, height of the lowest branch, and diameter at breast height (DBH). We measured these same characteristics for trees that were available to frogs, but not used by frogs during our study; we selected all unused trees within 3 m of the stream along one side of a 200-m section of stream for measurement. To determine whether frogs selected trees that were different from available trees, we analyzed our data using a two-way MANOVA. We used tree type (used or available) and site as independent variables, and tree height, height of the lowest branch, and DBH as dependent variables.3,27 = 3.192, P = 0.039), but not by site , and there was a marginally significant interaction between season and site . One-way ANOVAs on each variable indicated that the only significant univariate result was that frogs showed a seasonal difference in horizontal movements between day and night locations , although the seasonal difference in vertical movements between day and night locations was nearly significant . The overall pattern was that frogs moved significantly longer horizontal distances between diurnal shelter sites and nocturnal perch sites during summer than in winter at both sites . At Windin Creek, the length of stream used by frogs during summer was 78.9% longer than that used during winter, but at Frenchman Creek, the length of stream used did not differ significantly between seasons. The median stream length used by frogs at Windin Creek was 2.0 m (range: 0–16 m) during winter, and 9.5 m (range: 0–57 m) during summer, and the median (and range) across seasons was 4.0 m (0–50 m) at Frenchman Creek.The length of stream used by frogs during our three-week study period was influenced significantly by the interaction of season and site  and sites , and there was a significant interaction between season and site . Frog position relative to the stream during the night also differed significantly between seasons , but not between sites ; however, there was a significant interaction between season and site . Results from one-way ANOVAs indicated that frog perch sites were higher above the stream during summer than winter, and that the extent of this difference depended upon site; heights were similar at both sites during winter, but tended to be higher at Frenchman Creek than Windin Creek during summer during both the day , although there was a trend during the day ; frogs were observed farther from the stream during summer than winter, and this pattern did not differ between sites during the day  or night  differed significantly between seasons , and sites , and there was a significant interaction between season and site , and those that were above 25°C , but not those that were below 15°C . The proportion of temperatures below 15°C was higher during winter than during summer at both sites (B. dendrobatidis growth (15–25°C) during winter than summer, and they reached temperatures above this threshold (>25°C) rarely during winter, but frequently during summer  and by time of day during both winter  and summer  and sites , and there was a significant interaction between the effects of season and site . Evaporative water loss in nocturnal microhabitats differed significantly between seasons  and sites , but the interactions between these effects was not significant . Microhabitats used by frogs during the night were significantly drier during winter than summer, but diurnal microhabitats were significantly drier during summer than winter. Overall, microhabitats used by frogs during the day and night at Frenchman Creek were significantly drier than those used at Windin Creek.Evaporative water loss  in diurnal microhabitats used by frogs differed significantly between seasons , although this difference appears to be driven only by differences in water depth below perch sites . Additionally, there was a significant interaction between perch type (used or available) and site . The overall pattern was that, when compared to available vegetation, frogs selected vegetation that was significantly taller, had a significantly fuller canopy , and was located significantly closer to riffles and closer to the stream edge , and the difference between the average water depth below perch sites was marginally greater at Windin Creek than at Frenchman Creek . At Frenchman Creek, frogs used vegetation that was 1.3 m taller, on average, during summer than during winter, but at Windin Creek, average vegetation height was similar between the seasons (0.2 m difference). During summer, frogs often used trees as perch sites; the trees used by frogs were significantly different from available trees . Overall, the characteristics of trees present at the two sites differed significantly ; trees were taller  and larger in DBH  at Frenchman Creek than at Windin Creek. There was also a significant interaction between tree type (used or available) and site . Frogs at Frenchman Creek selected trees that were significantly shorter than available trees , whereas frogs at Windin Creek did not select trees that were significantly different in height than available trees . At both sites, frogs selected trees with significantly lower branches than those of available trees ; the average height of the lowest branch of used and available trees was 2.3 and 5.0 m, respectively, at Frenchman Creek, and 2.1 and 6.9 m, respectively, at Windin Creek. Frogs did not select trees that differed significantly in DBH  from available trees; on average, the DBH of used trees was 9.5 cm at Frenchman Creek, and 3.2 cm at Windin Creek.When we compared the height of vegetation used by frogs during winter and summer, we found a significant interaction between vegetation height and site (FLitoria rheocola), an IUCN Endangered species [L. rheocola are relatively sedentary frogs that are restricted to the stream environment, and prefer sections of the stream with riffles, numerous rocks, and overhanging vegetation  year-round. This temperature range is based on the performance of B. dendrobatidis cultures maintained at constant temperatures [Once a frog is exposed to a nfection ,19. Rateiccation , and temiccation ,12. Our ons Figs  and 4; teratures  and on feratures ,19,54.B. dendrobatidis, it is important to study the behavior of uninfected individuals because this pathogen may alter the behavior of amphibians [L. rheocola, and reveals how these behaviors differ by season and between sites varying in elevation. Seasonal differences in environmental conditions and frog behavior cause this species to be most vulnerable to B. dendrobatidis during cooler months and at higher elevations, providing an ecological mechanism for observed patterns of infection dynamics [Detailed ecological studies are necessary to understand and conserve endangered species. Even closely related frog species that occur at the same sites can have very different patterns of movement and microhabitat use, and can also differ considerably in their vulnerability to disease-related declines ,15,19. Fphibians –36. Our S1 File(XLS)Click here for additional data file.


SLC20A2-gene encoding the inorganic phosphate (Pi) transporter PiT2 can explain approximately 40 % of the familial cases of the rare neurodegenerative disorder primary familial brain calcification (Fahr’s disease). The disease characteristic, cerebrovascular-associated calcifications, is also present in Slc20a2-knockout (KO) mice. Little is known about the specific role(s) of PiT2 in the brain. Recent in vitro studies, however, suggest a role in regulation of the [Pi] in cerebrospinal fluid (CSF). We here show that Slc20a2-KO mice indeed have a high CSF [Pi] in agreement with a role of PiT2 in Pi export from the CSF. The implications in relation to disease mechanism are discussed.Mutations in the  SLC20A2, which encodes the type III Na+-dependent inorganic phosphate (NaPi) symporter PiT2  lower than its blood [Pi]  compared to wild-type (WT) mice.The choroid plexus (ChP) localized at the lateral, the third, and the fourth ventricles produces most of the CSF, which has a high daily turnover rate . In situouse ChP . Recentlood [Pi] . Their r of PiT2 , e.g., i of PiT2 . Only twral ChPs . These rTac-Slc20a2tm1a(EUCOMM)Wtsi/Ieg (EM:05549), were obtained from the European Mouse Mutant Archive, Germany; the strain has been described previously  in the CSF, we measured the [Pi] in CSF and blood drawn from 3-week-old Slc20a2-KO mice and WT litter mates. In average, the WT mice showed a serum [Pi] of 4.79 ± 0.65 mM (standard deviation) and a CSF [Pi] of 0.90 ± 0.25 mM    (Table Slc20a2-KO mice were unable to sustain their CSF [Pi] at the same low level as the WT mice.The average serum [Pi] of the SLC20A2 are linked to PFBC [Slc20a2-KO mice [Slc20a2-KO mice to sustain the same low CSF [Pi] as found in WT mice is in agreement with a role of PiT2 as an exporter of Pi from the CSF. The elevated CSF [Pi] likely also leads to an elevation of ISF [Pi]. With reference to hyperphosphatemia, we suggest an introduction of the term CSF hyperphosphate to describe Pi levels in the CSF above the highest concentration occurring in the non-pathologic situation.It is now well-established that deleterious mutations in the gene  to PFBC –17, whic to PFBC , 19. A s-KO mice  suggesteSLC20A2-associated PFBC has not been investigated, and it remains to be seen whether they also present with CSF hyperphosphate. The here presented new insight in the role of PiT2 in maintaining CSF normophosphate in mice, however, points at a potential mechanism, or contributing mechanism, behind the cerebral vessel disease present in SLC20A2-associated PFBC and sporadic cases, i.e., CSF hyperphosphate. Specifically, pericytes/vascular smooth muscle cells in the brain might react in a similar manner to CSF hyperphosphate, as the peripheral vascular cells are suggested to react to hyperphosphatemia  in individual carriers of phatemia , 23, i.eXPR1, were also found associated with PFBC [SLC20A2 encodes an importer of Pi into cells [XPR1 encodes a protein exporting Pi out of cells [Xpr1 in mouse ChP [XPR1 mutations could potentially also result in CSF hyperphosphate.Recently, mutations in another gene encoding a protein associated with cellular Pi homeostasis, ith PFBC . Howeverto cells , 39, XPRof cells . XPR1 prof cells . Interesouse ChP . Albeit ouse ChP , 41. Thu


Modern agriculture has created a demand for plant biotechnology products that provide durable resistance to insect pests, tolerance of herbicide applications for weed control, and agronomic traits tailored for specific geographies. These transgenic trait products require a modular and sequential multigene stacking platform that is supported by precise genome engineering technology. Designed nucleases have emerged as potent tools for creating targeted DNA double strand breaks (DSBs). Exogenously supplied donor DNA can repair the targeted DSB by a process known as gene targeting (GT), resulting in a desired modification of the target genome. The potential of GT technology has not been fully realized for trait deployment in agriculture, mainly because of inefficient transformation and plant regeneration systems in a majority of crop plants and genotypes. This challenge of transgene stacking in plants could be overcome by Intra-Genomic Homologous Recombination (IGHR) that converts independently segregating unlinked donor and target transgenic loci into a genetically linked molecular stack. The method requires stable integration of the donor DNA into the plant genome followed by intra-genomic mobilization. IGHR complements conventional breeding with genetic transformation and designed nucleases to provide a flexible transgene stacking and trait deployment platform. The Green Revolution in the 1960s combined advances in breeding and agricultural practice, and provided food security to millions of people . In addiGenetically modified (GM) trait technology in the mid-1990s made a major impact in meeting the world food demand and there has been a rapid adoption of the technology. These first generation trait products involved simple herbicide and insect traits that required introduction of a single gene. Control of the broad range of insect pests and weeds desired today requires multiple insect and herbicide tolerance genes . In addiDesigned nucleases have become a powerful gene targeting (GT) tool to create targeted DNA double strand breaks (DSBs) at specified genomic locations, which stimulate the cell’s DNA repair machinery leading to integration of exogenously supplied transgenes into a specified genomic site. While designed nuclease-mediated targeted mutagenesis is becoming routine in plants –9, site-DSBs can arise spontaneously, may be induced by ionizing radiation and chemicals, or recently by designed nucleases . GenTargeted DSB-induced NHEJ has been previously described for mutagenesis, deletions or imprecise insertions , 19, 20.Targeted DSBs stimulate the cell’s DNA repair machinery making the DSB site accessible to a donor transgene for site-specific integration. The DSBs however do not preclude the ectopic integration of a donor transgene elsewhere in the genome. In addition, the GT process requires efficient delivery of the donor molecule to the DSB site and the ability to regenerate whole plants from the cells with a precisely repaired targeted genomic site. Random integration of the donor transgene and an inefficient transformation method for the donor delivery therefore are two major challenges for the routine deployment of GT technology in crop plants. Positive selection for GT such that precise insertion of the donor complements the non-functional selectable marker in the target locus has been used to avoid random integration of the donor , 23, 24 Agrobacterium  wou wou27] wve cells –59 can cFuture biotech crops are projected to require multiple transgenes to confer resistance to a broad spectrum of insect pests and provide herbicide tolerance with different modes of action. Insects and weeds will eventually develop resistance, new target pests will emerge and new traits will inevitably be needed and desired, so designing those future products to be further modified and developing capabilities to accomplish the modifications are wise investments. It is clear that producing and modifying transgenic events through GT has many advantages over random integration, and technology continues to develop to make GT increasingly efficient and flexible. Intra genomic homologous recombination using designed nucleases has good potential to overcome limitations in plant transformation and breeding to achieve targeted and highly complex stacked trait crops.


The purpose of the study was to report a case of live birth following donor oocyte in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) in which the oocyte donor herself was conceived via IVF. To our knowledge, such a case has not been previously reported.Retrospective chart review; this case is reported after chart review of a successful outcome.A 42 year-old woman, with diminished ovarian reserve, and her husband desired to conceive. She underwent a fresh IVF/ICSI cycle with her own oocytes, which unfortunately was not fruitful in terms of pregnancy or cryopreserved embryos. The couple was counseled regarding the option of donor oocytes, and they elected to proceed with a fresh cycle of donor oocyte IVF/ICSI. The couple selected an anonymous oocyte donor from a donor agency who was a first-time oocyte donor and, interestingly, was conceived via IVF herself. The fresh donor oocyte/IVF/ICSI cycle did not result in pregnancy; however, two supernumerary blastocysts were cryopreserved for future cycles. The recipient’s subsequent frozen-thawed embryo transfer (FET) resulted in a singleton gestation and live birth.An oocyte donor who was conceived via IVF had good ovarian response to stimulation, a good number of oocytes retrieved, and the formation and cryopreservation of blastocysts which, in a subsequent FET cycle, resulted in pregnancy and live birth for a recipient couple. To our knowledge, this is the first case reported of live birth with the use of donor oocytes from an oocyte donor who herself was conceived via IVF. During the initial years of human clinical in vitro fertilization (IVF) , it had This case is reported after chart review of a successful outcome. Patient-informed consent was not requested since all patient data was de-identified. A computerized search was performed in PubMed and Medline databases. The keywords and various combinations of phrases used in the search included the following: “IVF offspring”, “IVF children”, “female fertility”, “female infertility”, “own fertility”, “women born from IVF”, “conceive”, “fertility”, “oocyte donor”, “egg donor”, “ART offspring”, and “ART children”. None of the encountered abstracts had reported the findings of this case report.A 42 year-old nulligravid woman, with diminished ovarian reserve, and her husband were interested in an IVF/ICSI cycle with donor oocytes after having undergone an autologous IVF/ICSI cycle with her own oocytes which resulted in a negative serum hCG level without surplus embryos for cryopreservation. They selected a first-time screened and tested 23 year-old anonymous oocyte donor who was available through an oocyte donation agency. The oocyte donor’s AMH level was 4.2 ng/ml. Interestingly, her medical history was significant for having been conceived via IVF. Her comments reflected having being raised by her parents to have a great appreciation for the IVF process and a desire to help women who cannot have children to experience the joy of doing so with assistance. She had previously delivered two children of her own which she had conceived without difficulty.The oocyte donor’s ovarian stimulation protocol consisted of an antagonist protocol with 300 IU of gonadotropins per day. After 11 days of ovarian stimulation, the oocyte donor’s peak estradiol level was 1369 pg/ml and her progesterone level was 0.8 ng/ml. Recombinant hCG (Ovidrel) was administered subcutaneously for final oocyte maturation, and 27 oocytes were retrieved 35 h later. Nineteen oocytes were M2 oocytes. ICSI was performed for the 19 mature oocytes, of which 11 fertilized and yielded 2PN zygotes and two yielded 3PN embryos; six mature oocytes did not fertilize. Ten of the 11 two pronuclear zygotes underwent cleavage and on day 3, two embryos, each graded 8A in accordance with the SART embryo grading system, were used for embryo transfer to the recipient’s uterus. In addition, two day 6 surplus blastocysts, each graded 3BB in accordance with the Gardner embryo scoring system, were cryopreserved by means of vitrification. The recipient unfortunately had a negative pregnancy test as a result of her fresh embryo transfer.The couple proceeded with frozen-thawed embryo transfer 4 months later. The wife’s endometrial preparation with an estradiol and progesterone protocol resulted in a trilaminar endometrium with a thickness of 9.0 mm. The two cryopreserved blastocysts were thawed and transferred to the patient’s uterus, resulting in pregnancy and live birth of a healthy singleton gestation.Since the initial years of IVF, many women who were conceived via assisted reproductive technologies (ART) have reached reproductive age and have had children of their own. Parallel to this progression has been a significant increase in the utilization of donor oocytes in recent years . Success


Its concentration increases in various pathologies. Increased values in cerebrospinal fluid (CSF) are ascribed to an activation of immune system. Using immunoturbidimetry, we examined concentrations of beta2-microglobulin in cerebrospinal fluid in a large group of 6274 patients with defined neurological diseases. Cell counts, total protein, albumin, glucose, lactic acid, immunoglobulins concentrations, and isofocusing (IEF) were also evaluated. We found substantial changes of CSF β2-m concentrations in purulent meningitis, leptomeningeal metastasis, viral meningitis/encephalitis, and neuroborreliosis, while in multiple sclerosis these changes were not significant. Intrathecal synthesis and immune activation were present in these clinical entities. A new normative study enables better understanding of beta2-microglobulin behavior in CSF.Beta2-Microglobulin ( Beta2-microglobulin is composed of 99 amino acids; due to one peptidic bound it creates a loop. It belongs to the immunoglobulin superfamily, and its primary and secondary structure is strikingly similar to IgG structure; thus, it is suggested to arise from the same ancestral gene [In 1968, Beggard and Bearn first isolated beta2-microglobulin  [β2-m increases in patients with kidney diseases, because 99% is excreted by the kidneys. β2-m can form long protein chains deposited in joints and tissues  [eukemia) –8. Serumoidosis) . urine beta2-microglobulin value indicates a damage of renal tubules because of the decrease of reabsorption. Measurement of concentrations in both serum and urine enables to differentiate a problem of cellular activation from a renal tubulointerstitial disorder [Increaseddisorder –16. cerebrospinal fluid is recommended within the enhanced level of examination [β2-m concentrations in CSF have been proposed as a reliable marker in different inflammatory, autoimmune, or neoplastic central nervous system disorders. Increased CSF values are attributed to an immune activation and lymphoid cell turnover, a production of tumorous cells, and releasing in tissue destruction in CNS.Cerebrospinal fluid (CSF) reflects changes occurring in brain and spinal cord. Unlike blood analysis, examination of cerebrospinal fluid is not disturbed by other organ systems influence, and the CSF compartment is comprehensively autonomous. Assessment of beta2-microglobulin inmination ; β2-m coβ2-m and ferritin in CSF was found as a useful instrument for differential diagnosis between viral meningitis and bacterial meningitis and for monitoring of ATB therapy effect [β2-m values in neonates with cytomegalovirus infection, by reason of an intrathecal synthesis [β2-m concentration correlates with a disease progression, and a decline of increased value reflects a successful therapy [CSF values of beta2-microglobulin were studied in the context with neurological diseases by many authors. Simultaneous measurement of y effect –22. Manyynthesis –25. Find therapy –31. Asse therapy –35.A new study shows an antibacterial role of beta2-microglobulin in further human fluid: the amniotic fluid .β2-m levels in cerebrospinal fluid is currently available. While most groups of patients described are numerous enough, what is badly needed is a comparison of diagnostical subgroups of defined neurological entities in reasonably large groups. We examined a large CSF sample and follow-up values of beta2-microglobulin in patients with defined CNS diseases to clarify a biological behavior of this marker. We hypothesize that CSF values of β2-m would be increased particularly in patients with CNS inflammations and tissue disintegration. Our results support the great importance of β2-m examination for differential diagnosis and early therapy of central nervous system diseases, with concordance with the other CSF parameters, incl. CSF cytology, and biochemistry.A large body of literature on monitoring β2-m in CSF and serum, and the isoelectric isofocusing were evaluated within an enhanced CSF protocol [In the Laboratory for CSF and Neuroimmunology of the Homolka Hospital  we evaluated a total of 6274 CSF samples from patients aged from 3 to 86 years. In all the cases, the CSF samples were analysed only for diagnostic purposes, and they were completely investigated. We examinated firstly a CSF cell count  and basic biochemistry including total protein, glucose, lactic acid concentrations, and CEB  . Secondlprotocol . With improtocol .Qalb only in the cases when albumin CSF and serum concentrations were measured simultaneously. Evaluation of a blood-CSF barrier permeability is not so important for β2-m as for other CSF proteins due to a small molecule of β2-m [β2-m is slightly lower than serum one and Qβ2-m is below 0.8 (see β2-m level and an equilibration of CSF and serum concentrations (whether any serum concentration), and consequently Qβ2-m elevated above 0.8. So we can say that only Qβ2-m increased above 1.0 points clearly to the intrathecal synthesis of beta2-microglobulin.The functional state of blood-CSF barrier could have been expressed by  of β2-m . Neverth 0.8 see . Then a β2-m were established using immunoturbidimetry in CSF and corresponding sera in Synchron LX-20 analyser. Cytological CSF syndromes with a normal cell count  were classified as normal or pathological oligocytosis  depending on the morphological and functional prevalence of individual cell lines in the cytological picture, whereas, with tumourous oligocytosis, the key factor was the detection of malignant elements in the cytological sample in general. In the case of pleocytosis, an elevated cell count in CSF, cytological syndromes were determined using similar criteria, that is, lymphocyte pleocytosis, monocyte pleocytosis, granulocyte pleocytosis, and tumorous pleocytosis, using classification acc. to Adam et al. [In cytological examination, we used standard and special staining techniques in an effort to detect malignant elements  and the presence of CNS tissue destruction (Oil Red 0) by detecting lipophagic macrophages. Cytological samples were obtained from CSF using a gentle sedimentation technique. Concentrations of m et al. . From thn = 2674), neuroborreliosis (n = 215), brain tumours/malignant infiltrations (n = 95), viral meningitis/encephalitis (n = 641), bacterial meningitis/encephalitis (n = 312), scavenger reaction (n = 638), and polyneuritis/polyneuropathies (n = 1261). Malignant meningeal infiltration (MMI) or meningeal carcinomatosis is defined as a meningeal metastatic involvement without the presence of lokalized metastases . This is relatively frequent severe complication of cancer, mostly in secondary, sometimes also in primary tumours of the nervous system. The phagocytosis of a specific substrate, that is, the scavenger reaction or reactions, is characterized by the presence of the phagocytosis of tissue destructive products and cellular detritus. This occurs in the presence of a morphological CNS and PNS impairment, for example, in brain ischemia or in CNS traumas [Control group was declared as the group with normal cytological and biochemical findings , and af traumas .t-test, and one-way ANOVA with pairwise post hoc multiple comparisons were used for statistical analysis. As the β2-m distribution is not normal, we performed the logarithmic transformation of β2-m for one-way ANOVA testing.The values were precisely statistically correlated, leading to substantial concrete findings both in the whole group of patients and after their division according to the presence of normal or pathological biochemical, and even cytological CSF findings. Descriptive methods, Kolmogorov-Smirnov test, box-and-whiskers plots, Results are summarized in Tables β2-m in cerebrospinal fluid are present in the pathological group. We found a statistically significant difference between the control group and the pathological group (P < 0.001). Conversely, serum β2-m values are similar in pathological and control group. Although β2-m concentration was not certainly used for determination of the control group, in this group the mean of β2-m is in the reference range both in cerebrospinal fluid and in serum. In the pathological group, the mean of β2-m is slightly increased and Qβ2 is increased only in CSF.As can be seen from β2-m in CSF and serum with individual diagnostic subgroups. We found the highest median of β2-m in cerebrospinal fluid in the group with bacterial meningitis/encephalitis, where increased β2-m values are surely caused by inflammatory response and an intrathecal synthesis . The second group is the group with malignant infiltrations or brain tumours; highly increased values of β2-m are probably caused by tissue destruction, production of tumorous cells, and inflammatory response as well (Qβ2 = 1.32). The third ones are equal groups with viral meningitis/encephalitis and scavenger reaction. We found borderline values of β2-mCSF in group with neuroborreliosis. Our results confirm that β2-m behaves in cerebrospinal fluid as an inflammatory marker or a tumour marker. Elevated β2-mCSF values should lead to a suspicion of CNS inflammation or malignant infiltration, even if no tumorous cells are present in cerebrospinal fluid. On the other hand we demonstrated only slightly or no increased values of CSF β2-m and no intrathecal synthesis  in groups with multiple sclerosis and polyneuritis/polyneuropathies. Concentrations of CSF β2-m probably reflect an activity and a progression of disease in patients with multiple sclerosis. In the control group, we found unique CSF and serum β2-m values greater than the upper reference limit  was not exceeded by medians in any diagnostic subgroup. Serum oups see .P < 0.0001), as well as in comparison with all other groups. In other words, beta2-microglobulin values are the highest in the group with bacterial meningitis/encephalitis  nor in laboratory evaluation. The etiology of these units is not usually completely clear, the majority of clinical case is autoimmune or metabolic one. An extensive inflammatory response is unusual in polyneuritis and polyneuropathies.β2-m values in groups with bacterial meningitis/encephalitis, malignant infiltrations or brain tumours, and viral meningitis/encephalitis. In these groups an immune activation or a tissue destruction is present. Our results confirm the role of beta2-microglobulin as an inflammatory or tumorous marker in cerebrospinal fluid. Further studies of beta2-microglobulin in children are needed to clarify a relation with a maturation of the blood-CSF barrier. And it is appropriate to deal with β2-m values in further clinical entities . Measurement of β2-m does not need any large costs; a requirement on the CSF amount is minimal. Our results support the great importance of β2-m examination for differential diagnosis, early therapy, and monitoring of therapeutic effect of central nervous system diseases.Today, laboratories determine, on a routine basis, many protein fractions in CSF and serum (or plasma). Terms like “CSF Proteinic Status” and “CSF proteinogram” appear. We examined a large CSF sample in patients with defined CNS diseases to clarify a biological behavior of beta2-microglobulin. The importance of our study is supported also by the fact that all measurements were performed in one laboratory by the same method of analysis. We found substantial elevation of


Handwashing is a cost-effective way of preventing communicable diseases such as respiratory and food-borne illnesses. However, handwashing rates are low in developing countries. Target 7C of the seventh Millennium Development Goals was to increase by half the proportion of people with sustainable access to safe drinking water and basic sanitation by 2015. Studies have found that better access to improved water sources and sanitation is associated with higher rates of handwashing.Our goal was to describe handwashing behaviour and identify the associated factors in Vietnamese households.Data from 12,000 households participating in the Vietnam Multiple Indicator Cluster Survey 2011 were used. The survey used a multistage sampling method to randomly select 100 clusters and 20 households per cluster. Self-administered questionnaires were used to collect data from a household representative. Demographic variables, the presence of a specific place for handwashing, soap and water, access to improved sanitation, and access to improved water sources were tested for association with handwashing behaviour in logistic regression.p<0.05). Those having access to an improved sanitation facility were more likely to practise handwashing , as were those with access to improved water sources .Almost 98% of households had a specific place for handwashing, and 85% had cleansing materials and water at such a place. The prevalence of handwashing in the sample was almost 85%. Educational level, ethnicity of the household head, and household wealth were factors associated with handwashing practice (Households with low education, low wealth, belonging to ethnic minorities, and with low access to improved sanitation facilities and water sources should be targeted for interventions implementing handwashing practice. In addition, the availability of soap and water at handwashing sites should be increased and practical teaching programs should be deployed in order to increase handwashing rates. Hands are not only an indispensable tool used for daily activities but also a vector for spreading infection. Washing hands is an effective strategy for preventing the spread of many diseases. Evidence shows that washing hands halves the risk of pneumonia and diarrhoea, which are two of the leading causes of deaths worldwide in children under 5 years old . A studyAlthough handwashing with soap and water is a simple and efficient method for reducing the risk of infectious diseases –7, a larHandwashing behaviour can be assessed using questionnaires, by handwashing demonstration, by direct observation, or by indirect observation. However, there is no consensus on a gold standard for identifying handwashing behaviour . Some arOne study found that soap and water are more likely to be available in households that have a specific place for handwashing . The avaNonetheless, there is little information regarding handwashing behaviour among the Vietnamese. One study showed a low rate of handwashing with soap in rural schoolchildren in northern Vietnam . In thatVietnam is a developing country, with 54 ethnic groups, of which the Kinh account for 86% of the total population of 86 million . VietnamThis study assessed handwashing behaviour and its associated factors using data from the Vietnam Multiple Indicator Cluster Survey (MICS), which is an ongoing survey that has been conducted by the General Statistics Office of Vietnam and the United Nations Children's Fund since 2000. The MICS collects information on children, women, and key indicators related to the Millennium Development Goals using a household questionnaire, which is administered to a household representative. The survey has now been conducted four times in Vietnam. However, MICS4 (2010–2011) was the only dataset used here because the surveys conducted in the other 3 years did not include data on handwashing behaviour .The sample size of 12,000 households was estimated and selected using a multistage sampling method, with clusters as the primary sampling unit (PSU). A systematic probability proportional to size sampling method was applied to select 100 clusters for each region, stratified by rural and urban areas. Random systematic sampling was applied to select 20 households in each cluster .Demographic information included the six Vietnamese regions, living areas, educational level, ethnicity, religion of the household head, and household wealth index. Other independent variables were the availability of an improved sanitation facility and improved water sources. The six Vietnamese regions were the Red River Delta; Northern Midland and Mountain Areas; North Central Area and Central Coastal Areas; Central Highlands; South East; and Mekong River Delta. Living areas were indicated as rural or urban. Educational levels of household heads were classified as none, primary school, junior high school, senior high school, and tertiary or higher level of education. Ethnicity was divided into Kinh/Hoa and other minority groups. Religion was grouped into Buddhism, Cao Dai, Hoa Hao, Catholic, Protestant, and other religion or no religion. A household wealth index was classified into five equal quintiles, with the first indicating the poorest and the fifth the richest. The wealth index (provided by the MICS) was derived using information on water source, toilet facility, housing, fuel types for cooking, electricity, bank account, durable goods , and animals  .improved sanitation facility was defined as ‘one that hygienically separates human excreta from human contact’ , tube well/borehole, protected well, protected spring, and rain water collection’ for drinking purposes. If bottled water was used, the households must have had to use any of the improved water sources listed above for other purposes such as cooking and washing hands, to be considered as using improved water sources  there was a specific place for washing hands; and 2) cleansing materials (i.e. soap) and water were available at that specific place. This was assessed by indirect observation when interviews with the household representative were conducted  for the i-th sample PSU in the h-th stratum is the probabilities of selection at every stage in each sampling stratum: fhi=p1hxp2hxp3h where pshi is the probability of selection of the sampling unit at stage s for the i-th sample PSU in the h-th sampling stratum’  having a specific place for handwashing, having water and cleansing materials, and practising handwashing were estimated by household characteristics.p-value set at 0.075 for removing a variable , household wealth index, improved sanitation facilities, and improved water sources, with handwashing behaviour as the dependent variable. The choice of independent variables was informed by previous findings reported in the literature , 21, 22.variable . Crude oAmong the 12,000 estimated households, 11,642 households were present at the time of the survey. The response rate was 99.8% . There were 28 cases with missing information on education and 2 cases with missing information on religion. Those records were excluded from the analysis.p=0.371).In The percentage of households living in urban areas was 29.7%. People living in urban areas were more than twice as likely to wash their hands compared to those who lived in rural areas .The Kinh and Hoa ethnic groups represented 89.9% of the study sample and were more likely to wash their hands compared to other minor ethnic groups . The households that used an improved sanitation facility were more than three times as likely to wash their hands compared to those that did not use an improved sanitation facility . The households that used improved water sources were more than three and a half times as likely to wash their hands compared with those that did not .p-value of 0.075 removed region, living area, and religion from the full model, as their p-values were high (>0.425). The final model included educational level and ethnicity of the household head, household wealth index, improved sanitation facility, and improved water sources (The multivariable logistic regression model identified factors associated with handwashing behaviours. Stepwise backward methods with a threshold  sources .p<0.001) compared to households where household heads had no education. Households where household heads had a tertiary or higher educational level were more than three times as likely to wash their hands  compared to households where household heads had no education.There was a positive association between educational level of household heads and handwashing. The higher the educational level, the more likely it was that household members washed their hands, and the odds gradually increased depending on educational level. For instance, if the household head had a primary school educational level, households were 1.66 times more likely to wash their hands , whereas people in the richest quintiles were more than three times  as likely to wash their hands.The household wealth index showed similar results. When comparing the poorest quintile to the rest of the quintiles, households in the second quintile were 1.6 times more likely to wash their hands . Households using improved sanitation facilities and improved water sources were also more likely to wash their hands . Moreover, when interactions were tested between improved sanitation facilities and improved water sources with handwashing behaviour, no statistically significant interaction was found .The Kinh and Hoa were almost one and a half times more likely to wash their hands compared to other minorities (aOR=1.45, 95% CI: 1.07–1.97, This study used data from the MICS to determine the prevalence of handwashing behaviour and identify the factors related to handwashing behaviour in Vietnam. Our findings showed that 84.7% of the households practised handwashing behaviour, which is similar to the prevalence of 80.8% reported in a previous study . Other fA study in Kenya showed that areas with a high density of Muslim people had a higher proportion of handwashing . In our Regarding the ethnic groups in Vietnam, the Kinh represent 86% and the Hoa represent 1% of the total population . As in tNevertheless, an evaluation of primary schoolchildren from minor ethnic groups stressed the importance of teaching programmes rather than simply investing in handwashing facilities such as water and soap . Thus, bA systematic review showed that hospital nurses can also educate and improve handwashing behaviours of patients, who will keep practising the learned behaviours even after being discharged . By the This study has some limitations. Although the MICS has been conducted every 5 years since 2000 in Vietnam, data on handwashing were collected for the first time in the MICS 2010–2011 and therefore it was not possible to estimate the trends in handwashing behaviour over time. Another limitation relates to the measurement of handwashing behaviour in the MICS. Indirect observation has its limitations, but it is still a reliable method for handwashing assessment , 11, 12.The strengths of our study are that the MICS is a large national survey that is representative of all regions in Vietnam. The MICS has been conducted for some years and the procedures have been tested and standardised to ensure high quality data are collected.The study showed that the overall percentage of handwashing was almost 85%. In order to promote handwashing behaviour, availability of soap and water at the sites for handwashing should be increased and, more importantly, practical teaching programmes should be deployed. Educational level and ethnicity of the household head, household wealth index, and access to improved sanitation facilities and improved water sources were all associated with handwashing behaviour. These factors should be taken into account when designing interventions to help ensure that resources are directed to those most in need, such as people living in disadvantaged areas and of low socio-economic status.


Cerebrospinal fluid (CSF) leak following spinal surgery is a relatively common surgical complication. A small group of CSF leak patients require multiple surgical repairs and prolonged hospital admission. Spinal CSF leaks are usually classically associated with symptoms of low intracranial pressure (ICP). However, there is a paucity of literature investigating the associated CSF dynamics.Retrospective case series study of patients with persistent CSF leak referred to the hydrocephalus service in our unit for intracranial pressure monitoring. Medical notes were reviewed for clinical presentation, management and outcome. Images were reviewed and ICP data were analysed. All patients underwent Continuous ICP monitoring using Spiegelberg ICP bolts.3 Patients had cervical, thoracic and lumbosacral complex spinal fixation surgery, complicated by prolonged CSF leaks (mean of 56 days from day of surgery to resolution). Each patient required 2 re-explorations spinal surgeries and multiple lumbar drains insertions prior to 24 hours ICP monitoring. Two patients were shown to have mildly raised ICP (>15.2mmHg) and all three had abnormal pulse amplitudes (>5mmHg). One patient underwent catheter cerebral venogram that demonstrated focal stenosis of the distal right transverse sinus with a significant pressure gradient. This patient underwent a right transverse venous sinus stent insertion. This resulted in resolution of headaches, prevention of impending wound breakdown and normalisation of ICP data. Two patients underwent insertion of ventriculo-peritoneal shunts (VPS). Both had resolution of their CSF leaks immediately post VPS insertion and were discharged from hospital.Our results suggest that abnormal cerebrospinal fluid dynamics should be explored in patients with persistent CSF leak post spinal surgery. Whether abnormal pressure and dynamics represent a pre-existing abnormality or is induced by spinal surgery should be subject for further studies.ICP: Intra Cranial Pressure; VPS: Ventriculo-peritoneal shunt.


Participants consumed two isoenergetic high-fat mealscomposed of a smoothie enriched with PO with v. without a cream-derivedcomplex milk lipid fraction ( dairy fraction rich in MFGM) separated by a washout of 1–2weeks. Serum cytokines, adhesion molecules, cortisol and markers of inflammation weremeasured at fasting, and at 1, 3 and 6 h postprandially. Glucose, insulin and lipidprofiles were analysed in plasma. Consumption of the PO + MFGM v. PO mealresulted in lower total cholesterol (P = 0·021), LDL-cholesterol(P = 0·046), soluble intracellular adhesion molecule(P = 0·005) and insulin (P = 0·005) incremental AUC, andincreased IL-10 (P = 0·013). Individuals with high baseline C-reactiveprotein (CRP) concentrations  had higher(P = 0·030) insulin at 1 h after the PO meal than individuals with CRPconcentrations <3 mg/l (n 19). The addition of MFGM attenuatedthis difference between CRP groups. The addition of a dairy fraction rich in MFGMattenuated the negative effects of a high-SFA meal by reducing postprandial cholesterol,inflammatory markers and insulin response in overweight and obese individuals,particularly in those with elevated CRP.Meals high in SFA, particularly palmitate, are associated with postprandial inflammationand insulin resistance. Milk fat globule membrane (MFGM) has anti-inflammatory propertiesthat may attenuate the negative effects of SFA-rich meals. Our objective was to examinethe postprandial metabolic and inflammatory response to a high-fat meal composed of palmoil (PO) compared with PO with an added dairy fraction rich in MFGM (PO+MFGM) inoverweight and obese men and women ( Postprandial inflammatory and lipaemic responses are pronounced inindividuals with obesity, the metabolic syndrome (MetS) and type 2 diabetes(–), in part because the magnitude of the postprandial inflammatory responseis correlated with insulin resistance. Current literature suggests that the inflammation of the postprandialstate adds to the already pro-inflammatory environment in individuals with obesity-inducedmetabolic disease, intensifying the overall systemic inflammation that underlies metabolicdysfunction. Given the rise in overweight and obesity worldwide, potential nutritional interventions that can limit the postprandialinflammatory response would be of great public health benefit.The postprandial state has been highlighted as an important transitory period whensignificant vascular damage can occur and has recently been implicated in the causal processesof CVD(–). It has been shown that SFA induce inflammationpostprandially. Palmitic acid in particular appears to be detrimental. The consumption ofpalmitic acid acutely increases the insulin response compared with oleic acid andn-3 fatty acids and this was shown to be related to the adverse effects of palmitate onboth β-cell function and insulin sensitivity in the postprandial state. Palmitate, either as an isolated fatty acid or as part of a high-SFAmeal, increases inflammatory markers in the postprandial state. Palm oil (PO) is enriched in palmitate and has been used widely in thefood industry as a substitute for trans-fatty acids, which are known to havedeleterious effects on CVD risk.The postprandial inflammatory response is influenced by the fat content and composition ofthe test mealin vitro and animal models(–). MFGM, a protein–lipid complex originating from the apical surface ofmammary epithelial cells, surrounds the fat globules in milk and is found in dairy products atvarying levels. Specific proteins and lipids of MFGM are associated with health-promotingbioactive functions. For example, one major MFGM-associated protein, lactadherin, was reportedto bind and neutralise viruses, reduce intestinal inflammation, improve intestinalpermeability, and repair intestinal epithelium. MFGM-derived polar lipids were reported to have bactericidal properties,bind enterotoxigenic pathogens and reduce intestinal inflammation. In addition to functions attributed to its individual components, MFGM asa complex also reduced inflammation in vitro, in animals andclinically. However, it is not known whether the addition of a dairy fraction rich inMFGM to meals high in saturated fat would blunt the postprandial inflammatory response inhuman subjects.Milk fat globule membrane (MFGM) fractions have previously been reported to reduceinflammation in The objective of this study was to determine the postprandial inflammatory effect of ahigh-saturated fat meal using PO with and without the addition of MFGM in overweight and obeseadults. We hypothesised that consuming a high-fat PO + MFGM meal would result in lowerpro-inflammatory serum markers compared with the isoenergetic PO meal.2) plus havetwo or more MetS traits according to the definition of the American Heart Association orsimply be obese according to their BMI (30–39·9 kg/m2) and have any number ofMetS traits. The MetS is defined by having three or more of the following traits: waistcircumference >40 inches (>102 cm) for men and 35 inches (>89 cm)inches for women; fasting plasma TAG ≥ 150 mg/dl (≥ 1·70 mmol/l); fasting plasmaHDL-cholesterol <40 mg/dl (<1·04 mmol/l) for men and <50 mg/dl(<1·30 mmol/l) for women; blood pressure ≥130/85 mmHg; and fasting glucose≥100 mg/dl (≥5·56 mmol/l). Individuals were excluded from participation for the followingreasons: diagnosis of immune-related diseases, gastrointestinal disorders, cancer, type 2diabetes, eating disorder, allergies to the provided study foods, poor vein accessibilityaccording to the research phlebotomist, or a body-weight change greater than 10 % over thepast 6 months. Individuals were excluded from participation if they used the following:weight loss medications; daily non-steroidal anti-inflammatory drugs (NSAID);anti-inflammatory supplements; corticoid steroids; tobacco; change in hormonal birthcontrol regimen with the past 6 months; initiation of statins in past 3 months. Because ofpossible confounding effects on inflammatory outcomes, dietary exclusion criteria were asfollows: >1 serving of fish/week; >14 g fibre/1000 kcal (4184 kJ) per d;<16:1 of total dietary n-6:n-3 ratio; >1 %of daily energy as trans-fats; and a vegetarian diet pattern. Ifindividuals had initiated an exercise programme within the past 6 months, planned tobecome pregnant within the next 6 months, or were already pregnant or lactating, they werenot enrolled in the study. To determine enrolment eligibility, questionnaires wereadministered regarding health history, diet and medication. An online FFQ was used toassess dietary intake and a fasting blood sample was drawn for the analysis of bloodlipids and glucose. Additional anthropometric measurements were taken during the screeningvisit to determine MetS traits .A total of seventeen adult men and nineteen adult women  were recruited from the Davis and greater Sacramento areas of California toparticipate in this study. To qualify, individuals had to be between 18 and 65 years ofage, and either be overweight according to their BMI . Participants were assigned to testmeal order using a random number generator which randomly returned either a 0 or 1, with a0 being assigned to PO first followed by PO + MFGM second, and a 1 being assigned toPO + MFGM first followed by PO second. Test meals were consumed in random order ondifferent test days separated by a washout phase of minimally 1 week and maximally 2 weeksto avoid any carry-over effects. After each washout, participants consumed the alternatetest meal.To limit confounding effects, the consumption of anti-inflammatory supplements, alcoholor NSAID was not permitted for 72 h before each test day. At 24 h prior to the test day,vigorous exercise was prohibited to avoid increasing inflammatory markers and converselyconsumption of seafood was not allowed to avoid a suppression of inflammatory markers. Toensure compliance, participants filled out a 1-d food record for the 24 h prior to eachtest day. The dietary records were analysed using the Nutrition Data System for Research.. A fasted blood sample was drawn via venepuncture. Blood pressure,heart rate, weight and waist circumference were measured. The dietary test meal was thenconsumed completely within 20 min. Postprandial blood draws were conducted at 1, 3 and6 h. These time points were determined based on previous postprandial clinical trialsobserving a peak in pro-inflammatory cytokine concentrations around 3–6 h after consuminga high-fat meal.Participants arrived at the Western Human Nutrition Research Center after a 10–12 h faston each test day. The 24 h diet record was collected and participants were asked tocomplete a modified gastrointestinal questionnaireConsumption of any food other than the test meal was not permitted, but bottled water wasoffered throughout the test day. Participants were offered the option to stay at theresearch facility or leave between blood draws via car to limit physical activity and hadto return 15 min before their scheduled blood draw to allow for a 10 min rest period priorto each blood draw.. BPC50 is comprised of the following (% w/w): 52 % protein of which13·2 % is membrane-derived protein, 6·6 % lactose and 36·2 % total fat (22·5 % TAG and13·7 % phospholipids), 0·63 % gangliosides (GD3), and 5·2 % ash(–). The six highest abundant MFGM-derived proteins reported in BPC50include: fatty acid-binding protein, butyrophilin, lactadherin, adipophilin, xanthineoxidase and mucin. The PO smoothie contained whey protein isolate to match the proteincontent found in the BPC50 product. For ingredient details, see Supplementary Table S1.Participants were instructed to eat the entire meal, rinse their cup with water, and drinkthe rinse-water.The two test meals were made up of a bagel with strawberry preserves along with either aPO or PO + MFGM smoothie. In each instance the smoothie consisted of deionised water,cream of tartar, PO shortening, and raspberry sorbet. Additionally, the PO + MFGM smoothiecontained BPC50, a cream-derived complex milk lipid fraction powder (β serum concentrate)that is a proprietary product supplied by Fonterra Co-operative Group Ltd . To determine each participant's physical activity level the BaeckePhysical Activity questionnaire was used.Each test meal provided 40 % of the participant's total daily energy intake. Energyintake was determined by using the National Academy of Sciences equation from theInstitute of Medicine Dietary Reference IntakeThe two isoenergetic test meals were constructed to vary less than 0·2 % inmacronutrients and provided about 55 % fat, about 30 % carbohydrates, and about 15 %protein. Each test meal provided between 49 and 87 g of fat depending on each individual'senergy intake, 61–107 g of carbohydrates, and 31–55 g of protein . Test meg at 4°C for 10 min. EDTA-whole blood tubes were kept on ice during and after bloodcollection and were centrifuged within 30 min of collection at 1300 g at 4°C for 10 min. After centrifugation, the serum and plasma tubes were kept onice during aliquoting. Subsequently, plasma and serum aliquots were directly frozen at–70°C until analysed.Whole blood was drawn at baseline, and at 1, 3 and 6 h after the meal. Serum tubes wereallowed to clot at room temperature for 30 min, and then centrifuged at 1300 Serum samples from all four time points were analysed for cytokine concentrations , as well as thevascular injury molecules C-reactive protein (CRP), serum amyloid A, soluble intracellularadhesion molecule (sICAM) and soluble vascular adhesion molecule. Plasma was used tomeasure IL-18 concentrations. A commercially available Multi Spot ELISA kit was used toquantify the concentrations of these markers .The protocol was followed as recommended by the manufacturer. Briefly, pre-coated plateswith capture antibodies were incubated with 25–50 µl of serum or plasma. After washing theplates a labelled detection antibody was added. Upon electrochemical stimulation the bounddetection antibodies emit light, which is measured by the plate reader to quantify theamount of each protein of interest.Serum cortisol was measured at all times points using the DetectX Cortisol EnzymeImmunoassay kit (Arbor Assays). Briefly, a cortisol–peroxidase conjugate and a monoclonalcortisol antibody were added to a pre-coated ninety-six-well plate. Upon incubation, serumsamples were added to each well and allowed to bind with the cortisol–peroxidaseconjugate. The total amount of cortisol present in each sample was then calculated basedon the absorbance detected by the reader.At each time point plasma glucose, insulin, and a lipid panel including TAG, totalcholesterol, HDL-cholesterol, LDL-cholesterol, HDL:LDL ratio, and non-HDL-cholesterol wereassessed by standard clinical techniques in the clinical laboratory of University ofCalifornia Medical Center .Body weight, height, waist circumference, blood pressure and heart rate were measured oneach test day. Body weight was measured with a calibrated scale . Waist circumference was measured in the standing position with measurementsmidway between the lateral lower rib margin and the ileac crest . Height was measured with a wall-mounted stadiometer . Blood pressure and resting heart rate were taken in theupright seated position using the appropriately sized cuff . Total fat mass andlean mass were assessed using dual-energy X-ray absorptiometry .. To ensure 95 % confidence of the results and 80 % power the samplesize calculation indicated that thirty-six participants would be needed.The sample size was calculated based on the primary outcome marker IL-6 using the meansand standard deviations from a similar human study with overweight men at risk fordeveloping the MetSP < 0·05. Normality wasestablished visually and numerically using histograms, Q–Q plots and the Shapiro–Wilktest. Data were transformed as needed. When concentrations for markers were below thelower limit of detection (LLOD)  for <25 % of thesamples, the value was calculated as the LLOD divided by 10. When concentrations formarkers were below the LLOD for >25 % of the samples, the data were excluded fromstatistical analyses (IL-1β and IL-4). Cases with values more than three box lengths fromthe 75th percentile or 25th percentile were deemed outliers and removed from all analyses;this only applied to sICAM where two subjects were excluded.Statistical analyses were conducted on SPSS version 20.0 software for Macintosh (SPSS).Differences were considered significant at t test was used. A mixed linear model wasperformed with treatment and time as fixed factors, participants as the random effect andtreatment × time as the interaction term. If time was significant, multiple-comparisonpost hoc analysis with Bonferroni correction was carried out to comparethe concentrations at 0–1 h, 0–3 h, 0–6 h, 1–3 h, 1–6 h, and 3–6 h.To determine if dietary differences existed between test meal composition and baselineanalyte concentrations a paired . The iAUC was chosen over the total AUC because it reflects thepostprandial rise of these metabolite concentrations above the non-zero fastingvalue. iAUC between test meals were compared by one-way ANOVA.The incremental AUC  and decremental (area below baseline)using the conventional trapezoid method were used to compare postprandial responsesbetween test mealsn 17), lowCRP was defined as <3 mg/l (n 19). Baseline characteristics of each CRP group can be found in To determine if pre-existing clinical conditions affected the inflammatory responses tothe test meals, secondary analyses were conducted. Participants were coded as having highor low CRP levels based on their baseline levels prior to receiving either test mealtreatment. High CRP was defined as a concentration ≥3 mg/l , and MFGM,which is enriched in medium-chain SFA characteristic of dairy fat.Participants consumed two test meals, a high-fat PO test meal and a high-fat PO + MFGMtest meal. The meals  were isoP = 0·04), HDL-cholesterol (P = 0·01), TAG(P < 0·0005), non-HDL-cholesterol (P = 0·04) andinsulin (P < 0·0005)  . Among tP = 0·02 withall subjects included, P = 0·04 with two outliers removed),LDL-cholesterol (P = 0·046) and a significantly higher concentration ofTAG (P = 0·025) when compared with the PO meal alone .The total concentration of each analyte over the 6 h postprandial time was calculated asthe iAUC and compared between test meals. The addition of MFGM to the test meal resultedin significantly lower concentrations of total cholesterol (P = 0·005) (P < 0·0005) that was complemented by a decrease inglucose (P < 0·0005). From 1 to 3 h there was an increase back tobaseline levels for glucose (P = 0·003) and a corresponding decrease ininsulin concentrations (P < 0·0005) although still greater thanthe 0 h value (P < 0·0005). The increase in insulin concentrationwas dampened from 0 to 1 h by more than 50 % when MFGM was added to the test meal,resulting in a 482 % increase v. a 234 % increase for POv. PO + MFGM, respectively . When total insulin concentration was compared over the 6 h postprandial period (i.e. theiAUC of insulin from 0 to 6 h) the addition of MFGM resulted in a significantly lowerexposure to insulin (= 0·005) . Neitherv. low baseline CRP concentrations, additional secondary analyses wereconducted. Participants with baseline CRP concentration ≥3 mg/l (coded as ‘high’) hadsignificantly higher insulin concentrations at 1 h after consuming the PO meal(P = 0·03). The addition of MFGM to the meal suppressed the insulinresponse in the high CRP group, thus removing any significant difference between the highand low CRP groups .The two cytokines IL-1β and IL-4 fell below the detection limit for 70 and 95 % ofsamples, respectively. Consequently, these markers were not included in the statisticalanalyses reported here. Similar results for IL-1β and IL-4 have been observed in previousstudiesP = 0·013) and sICAM was significantly lower in response to thePO + MFGM test meal  (P = 0·03), IL-8 (P = 0·04) and sICAM(P = 0·02)  following the PO meal, but IL-8 was unchangedafter the PO + MFGM meal. Concentrations for sICAM significantly increased from 0–1 h,0–6 h, and 3–6 h and significantly decreased between the 1–3 h time points after consumption of the PO meal and, likeIL-8, was unchanged after the PO + MFGM challenge. There was no treatment effect observedfor any of the other inflammatory markers, but a significant change over time was observedfor IL-6, IL-8, TNFα, CRP, serum amyloid A, sICAM, soluble vascular adhesion molecule andcortisol   . An inte = 0·02) . Over tifor all) . There wP < 0·05 for each timepoint) between participants with high v. low baseline CRP concentrationsafter consuming the PO test meal. After consuming the PO + MFGM test meal, this differencewas no longer significant, suggesting that MFGM may attenuate the postprandialinflammatory response in individuals with high CRP levels.Secondary analysis using baseline CRP as a covariate revealed a statistically significantdifference for IL-6 at each time point  over the course of each test day.Decreases throughout the day, from baseline to 1 h, 1–3 h, and 3–6 h were as follows: 15,33 and 43 %, respectively. This observed decrease throughout the day is consistent withdiurnal patterns of cortisol.Analysis of serum cortisol revealed no time × treatment interaction effect or a treatmenteffect. There was a significant decrease in cortisol concentration for all time points. In our study the total amount of sICAM over the 6 h postprandial periodin response to PO + MFGM was significantly lower by 95 % compared with PO. These resultssuggest that the addition of a dairy fraction rich in MFGM attenuates the atherogenic milieutriggered by the PO meal.Cellular adhesion molecules, such as sICAM, are key players in the early events ofatherosclerosis developmentv. PO + MFGMtreatments could stem from the difference in the fatty acid composition. The PO + MFGM mealwas higher in short- and medium-chained SFA and lower in 18 : 2n-6 and 18 :1n-9 compared with the PO meal. Chen etal. showed that when human retinal vascular endothelial cells were treatedwith linoleic acid (18 : 2n-6) it resulted in increased ICAM expression.The MFGM preparation used is composed of complex lipids including sphingolipids, as well asbioactive proteins, which may also play roles in the observed anti-inflammatoryeffects.It is possible that the difference in sICAM between the PO . Branched-chain amino acids may promote insulinsecretion; thus the effect of MFGM on insulin may be related to its amino acidcomposition.When analysing the insulin iAUC over the 6 h postprandial period, the total insulinconcentration was significantly lower in response to the PO + MFGM compared with the PO testmeal. To our knowledge there have not been any prior clinical trials examining the effect ofMFGM consumption on the insulin response in human subjects. One study investigated thepostprandial effect of adding a dairy product rich in sphingolipids, a lipid constituent ofMFGM, to a high-fat breakfast meal and found no significant difference in postprandialinsulin concentrationsThe secondary analysis based on baseline CRP concentrations revealed that participants whowere in an inflamed state (CRP ≥ 3 mg/l) in the fasted condition had significantly higherinsulin concentrations after consuming the PO meal compared with those who had normalbaseline CRP concentrations. However, the addition of MFGM to the high-fat test mealcompletely removed this difference. These results suggest an interaction between diet andphenotype, whereby consumption of MFGM by chronically inflamed individuals normalisedresponses to a high-fat meal to closely resemble that of a metabolically healthy profile., which was reflected in the present study, and decreases over the courseof the day. Elevated levels of cortisol inhibit the synthesis of pro-inflammatorycytokines. We hypothesised that this potential inhibition may explain the observedinitial decrease in the pro-inflammatory cytokines  from baseline (blooddraw schedule between 08·00 and 09·00 hours) to the 3 h blood draw (scheduled between 11·00and 12·00 hours). However, none of the correlations was significant.Research has shown that cortisol peaks in the morning53, whicIn summary, the addition of a dairy fraction rich in MFGM reduced the iAUC in postprandialinsulin, total cholesterol, LDL-cholesterol and sICAM responses over the 6 h postprandialperiod, and increased the production of the anti-inflammatory cytokine IL-10. The additionof a dairy fraction rich in MFGM also attenuated the increases in insulin at 1 h inindividuals with elevated fasting CRP. Results from this study suggest that the addition ofa dairy fraction rich in MFGM attenuates the negative metabolic and inflammatory effects ofa high-fat meal rich in saturated fat, specifically palmitate.


Atrioventricular (AV) groove disruption is a lethal complication of mitral surgery. Traditional teaching mandates that an "internal" repair be performed requiring prosthesis explantation, complete atrioventricular groove reconstruction utilizing a patch positioned from within the cardiac cavity, and subsequent prosthesis re-implantation. This is a massive undertaking and yields uniformly poor results.We examine the utility of an alternative, "external" approach for rescue of atrioventricular groove disruption.The study was conducted as a retrospective review of a multi-surgeon, multi-institutional experience inclusive of all patients suffering disruption of the atrioventricular groove following mitral surgery. All patients experiencing this complication (consecutively and exclusively) underwent "external" repair as a rescue procedure. The external repair technique was conducted on cardiopulmonary bypass support using direct (felt pledget/strip reinforced) suturing of the AV groove. This was supplemented as needed with applications of BioGlue, external bovine patch, left atrial patch suture technique, and coronary bypass of the circumflex system.Over a span of 20 years, 3071 mitral valve surgeries resulted in 13 AV groove disruptions (incidence 0.42%). Average patient age was 75.2 years (range 59-90), 77% (10/13) were female. Thirty-day mortality was 15.4% (2/13). Hospital mortality was 23.1% (3/13). Hospital stay (day of surgery to day of discharge) averaged 27.6 (range 8-42) days. Patient follow-up averaged 5 years (range 3 months to 10 years). One-, three-, and five-year survival rates were 72.7%, 72.7%, and 44.4%. Seven patients are currently still alive and remain in (no greater than) NYHA Heart Failure Class II, with an average follow-up of 4 years (range 3 months to 8 years). One patient developed a large chronic pseudoaneurysm originating from the AV groove repair site identified after discharge as an outpatient.The described "external" approach represents an effective, alternative repair technique for rescue of otherwise lethal atrioventricular groove disruption.


The interplay between the novel adipokine retinol-binding protein-4 (RBP4) and coronary artery disease (CAD) is still obscure. We investigated the relationship between RBP4 levels and the presence and severity of angiographically proven CAD and determined its possible role in acute myocardial infarction (AMI).305 individuals with angiographically proven CAD (CAD-patients), were classified into 2 subgroups: 1) acute myocardial infarction , and 2) stable angina . Ninety-one age- and sex-matched individuals without CAD, but with at least 2 classical cardiovascular risk factors, served as controls (non-CAD group). RBP4 serum levels were measured at hospital admission and were analyzed in relation to the coronary severity stenosis, assessed by the Gensini-score and the number of coronary narrowed vessels. Other clinical parameters, including insulin levels, HOMA-IR, hsCRP, glycaemic and lipid profile, and left-ventricular ejection fraction were also assessed.2 = 0.422, p < 0.001). Similarly, RBP4 concentrations increased with the number of coronary narrowed vessels (p < 0.05).Serum RBP4 levels were significantly elevated in patients with CAD compared to non-CAD patients . We did not observe a significant difference in RBP4 levels between AMI and SA subgroups (p = 0.734). Logistic regression analysis revealed an independent association of CAD presence with serum RBP4 , and hsCRP  levels, in the whole study group. Among variables, hsCRP (β = 0.220), HDL (β = −0.150), and RBP4 (β = 0.297), correlated in both univariate and multivariate analysis with CAD severity (RPatients with CAD, both SA and AMI, showed elevated RBP4 serum levels. Notably, increased RBP4 concentration seemed to independently correlate with CAD severity, but no with AMI.NCT00636766The ClinicalTrials.gov Identifier is:  Coronary artery disease (CAD) is the leading cause of morbidity and mortality worldwide with the risk for CAD development being higher among patients with obesity, diabetes and other insulin resistant states. Numerous studies have been previously conducted in order to identify novel biomarkers indicating either the presence or the severity of CAD ,2. Such A growing body of evidence, the adipocyte-derived cytokines, known as adipokines, seem to interfere with the crosstalk between adipose tissue, insulin resistance and CAD . RetinolAccumulating data strongly support the association of RBP4 circulating levels with traditional,  and non-traditional cardiovascular risk factors (e.g. cytokines) mainly through the impairment of glucose and lipid metabolism and adipose tissue dysfunction, despite that opposite findings put RBP4 changes in dispute –14. AlthIn this study, we sought to investigate the relationship between RBP4 levels and the presence and severity of angiographically proven CAD, and to determine its possible role in acute myocardial infarction (AMI).AMI (n = 141): Patients consecutively hospitalized in the coronary care unit of our department with acute myocardial infarction (AMI)  within 12 hours of symptoms onset. The AMI diagnosis was made on the basis of typical symptoms consistent with myocardial ischemia  that continued for >30 min, newly developed ischemic ST-T changes (ST-elevation or ST-segment depression or prominent T-wave inversion) in at least 2 contiguous ECG leads, and elevated cardiac-associated biomarkers of necrosis in an appropriate clinical setting.SA (n = 164): Consecutive patients with stable angina (SA) diagnosed as: chest pain or angina-equivalent on exertion, signs of myocardial ischemia on functional testing  and established CAD defined as previous myocardial infarction or percutaneous coronary intervention or coronary artery bypass graft. We excluded patients with unstable angina, such as those with recent onset of cardiac-origin symptoms or angina at rest and normal levels of cardiac troponin.We initially identified 868 individuals who underwent coronary angiography from January to June 2012 in “Attikon” University Hospital of Athens, Greece. We then excluded patients with history of recent, −within the past 6 months- severe chronic heart failure (class NYHA II - IV), malignant diseases, major trauma or surgery, severe renal (creatinine > 2 mg/dl) or liver insufficiency , acute or chronic infectious disease, or any kind of immune-mediated disease. Among individuals undergoing coronary angiography, 305 patients with significant, angiographically proven CAD, defined as stenosis of more than 50% of the luminal diameter in a major epicardial coronary vessel, were assigned to the CAD-group. Based on clinical and laboratory findings, the latter group was further subdivided into the following subgroups:Among the remaining individuals without angiographically proven CAD, we selected a cohort of 91 individuals, age- and sex-matched to CAD group. That group had at least 2 cardiovascular risk factors , but no clinical evidence of CAD, and served as control (non-CAD group). The main purpose to set such selection criteria in the control group was to limit confounders and create more comparable groups.The medical history of all participants was comprehensively recorded, and a clinical examination, electrocardiogram (ECG), trans-thoracic echocardiography and blood sampling, were carried out before angiography. The study complied with the Declaration of Helsinki, and the trial protocol was approved by the local Ethics Committee. Written informed consent was obtained from all patients before entering the study.2), waist-hip ratio (WHR), blood pressure, were determined. Waist circumference was assessed at the level midway between the lower rib margin and the iliac crest. The hips were measured at the level of the greater femoral trochanters. Thus, WHR expressed waist circumference divided by hip girth. BP was measured twice, after keeping all participants in a sitting position for 15 minutes. There was a 5 minute interval between the two measurements and the mean value was estimated for study purposes. Medications, co-morbidities and smoking history were assessed through a structured questionnaire preceding clinical examination. Before angiography, we also obtained the patients’ regular prescribed medications, based on medical records of the attending physicians. Diabetes was considered present if a patient was treated with insulin or oral agents or had a fasting glucose level ≥126 mg/dl (7.0 mmol/L), HbA1c ≥6.5% or a known history of diabetes. Hypertension was defined by systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg, the current use of antihypertensive treatment, or a combination of the 3. Dyslipidemia was defined as non-HDL levels ≥190 mg/dl, the current use of lipid-lowering treatment, or both [At the time of a clinical examination, body mass index  / height (m) or both . EchocarAll participants underwent coronary angiography by experienced cardiologists. Coronary artery severity was assessed using the number of narrowed vessels (>50%) Gensini score, which is derived from the co-evaluation of the number of stenotic coronary artery segments, the degree of their lumen stenosis and the localization of stenotic change . The GenIn patients with AMI, blood samples were drawn on admission and before treatment initiation to assess inflammatory markers. The rest of biochemical parameters, such as lipids and glycaemic indexes  were assayed after an overnight fast at least 24 h after admission. Similarly, venous blood samples were collected following an overnight fasting, in the rest of groups (SA and non-CAD). Glucose and lipids were analysed enzymatically . Low density lipoprotein cholesterol (LDL-C) was calculated by Friedewald formula. The intra-assay coefficients of variation (CVs) for total cholesterol, triglycerides, HDL and FPG were: 0.84%, 0.4%, 1.57% and 0.6%, respectively. In parallel, the inter-assay CVs for the abovementioned parameters were 1.3%, 1.3%, 2.00%, and 1.6%, respectively. The HbA1c was determined by high-performance liquid chromatography  in the diabetic subpopulation. A white blood cells (WBC) count analysis was performed using Cell Dyne 1700 electronic counter , with 1.99% intra-assay CV and 1.92% inter-assay CV.To measure insulin and RBP4, serum was separated from the blood corpuscles by centrifugation at 5000 g for 10 min and kept frozen at −80°C until the analysis. We quantified serum insulin with IRMA kit . The inter- and intra-assay CVs were 6.3% and 2%, respectively. Insulin resistance was calculated by a homeostasis-model-assessment (HOMA-IR) index with the following formula: HOMA-IR = fasting insulin (mU/L) × FPG (mg/dl)/405. Using a commercially available enzyme immunoassay kit, we assayed serum concentrations of RBP4 . The intra-assay CV was 9.7% and the inter-assay CV was 5%. Measurement of high-sensitivity CRP (hsCRP) was performed using a particle enhanced immunoturbidimetric assay . The detection limit was 0.1 mg/L, with intra- and inter-assay CVs of 1.34% and 5.7%, respectively. All serum concentrations (triplicate determinations) were analysed in a blinded manner with respect to any clinical information.Results are presented as mean values ± standard deviation (SD). Normality of distribution was assessed with Kolmogorov-Smirnov test. We used student’s t-test and chi-square for comparison of parametric and non-parametric variables between CAD versus non-CAD group and AMI versus SA subgroup. Comparison across all cohorts  was performed by one-way ANOVA and post-hoc Tuckey analysis. The relationships of RBP4 with other variables and CAD severity indexes were evaluated with Pearson correlation and standard multiple regression analysis. We explored the independent determinants of CAD in the whole study group using logistic regression analysis and the receiver operating characteristic (ROC) curve. A two-tailed p value <0.05 was considered to be statistically significant. We used the computer statistical software package SPSS .The clinical and laboratory characteristics of the whole study cohort and CAD subgroups are presented in Tables The AMI subgroup showed higher smoking rate (p < 0.001), SBP (p = 0.018) and DBP (p = 0.007) levels compared to SA subgroup. Regarding medications, we observed lower usage of statins (p < 0.001) and antiplatelet agents (p < 0.001) in AMI that SA subgroup before angiography. Notably, no patient was receiving vitamin supplements, which might have affected our results. As long as it concerns biochemical parameters, AMI-patients appeared with worse lipid profile and increased levels of FPG, HbA1c, WBC and hsCRP than the SA subgroup (p < 0.05).Significantly higher RBP4 values were found in CAD rather than non-CAD patients p < 0.001). Importantly, RBP4 concentrations did not differ between AMI and SA subgroups (p = 0.734). However, both AMI and SA subgroups showed considerably upregulated RBP4 concentrations than non-CAD group  .We searched for correlations of RBP4 with the rest of variables within the CAD group. In univariate analysis, RBP4 significantly correlated with hsCRP, fasting insulin and HOMA-IR. All those correlations remained significant in multivariate analysis  . In addition to this, RBP4 levels significantly increased across the number of diseased vessels from 34.19 ± 17.3 mg/L (1-vessel group) to 41.56 ± 16.32 mg/L (2-vessels group) and 49.19 ± 17.3 mg/L (3-/or 4-vessels group) (p < 0.05). In case of hsCRP, we found significant difference between 1-vessel and 3-/or 4-vessels group . On the other hand, HDL levels did not significantly change across the number of narrowed vessels.In univariate analysis, the severity of CAD, quantified by the Gensini score, was significantly correlated with FPG, HOMA-IR, hsCRP, HDL and RBP4 (p < 0.05). The latter variables entered standard multiple regression analysis Table . Among vMultiple logistic regression analysis was performed in the whole study group to estimate the association of significant CAD presence with clinical and biochemical variables, after adjustment for traditional cardiovascular risk factors like diabetes, hypertension, dyslipidemia, smoking and male gender. The presence of significant CAD was independently associated with serum RBP4 , and hsCRP  levels. The ROC curves of RBP4 and hsCRP for the discrimination between CAD or not are shown in Figure The present cross-sectional study demonstrated increased serum RBP4 levels in patients with CAD (either AMI or SA) compared to age- and sex-matched individuals without CAD, but with at least 2 classical cardiovascular risk factors. In our study cohort, the presence of significant CAD was independently related to RBP4 and hsCRP circulating levels. This is the first study demonstrating the independent association of RBP4 with CAD severity indexes (e.g. Gensini score and number of narrowed vessels).In spite of the rather established relation to insulin resistance and cardiovascular risk factors, the actual association of RBP4 levels with atherosclerotic-related cardiovascular disease is still controversial. Previous studies have shown the positive association of RBP4 with subclinical atherosclerosis –21. In eAccumulating data support the relationship of novel adipokines with CAD severity . To our in vitro inflammation in endothelial cells, by stimulating expression of proinflammatory molecules, such as vascular cell adhesion molecule 1 (VCAM-1), E-selectin, intercellular adhesion molecule 1 (ICAM-1), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6) [Regarding the underlying mechanisms, we observed the independent correlation of RBP4 with insulin resistance indices and established markers of inflammation, like hsCRP. More recently, RBP4 levels independently predicted early endothelial dysfunction, linking adipose tissue inflammation and subclinical atherosclerosis in non-diabetic individuals . The ass6 (IL-6) . Those e6 (IL-6) .The role of retinoids in lipid metabolism is well known and is mediated through the regulation of ApoC-III and VLDL production and fatty acid oxidation . A relatThe major limitation of the present investigation was the cross-sectional design, which prevented us from inferring cause-effect relationship of RBP4 with CAD. Although we did not recognise differences between acute and stable condition of CAD, the cross-sectional design of our study did not allow us to evaluate the association of RBP4 with either AMI occurrence or long-term clinical outcomes. Since the majority of patients with classical cardiovascular risk factors  were already treated, we cannot rule out the plausible effects of pharmaceutical agents (e.g. statins) on RBP4, leading to underestimation of its predictive power. Another important limitation was the considerable differences in some biochemical parameters between CAD and non-CAD groups, which might have affected RBP4 fluctuations. Despite the independent association between RBP4CAD and CAD diagnosis, the absence of matching for baseline characteristics may weaken our conclusions. Finally, as our control group sample comprised of patients with cardiovascular risk factors, we couldn’t extrapolate our conclusions to healthy subjects. Another study limitation is the potential influence of the transthyretin TTR-RBP4 complex in the affinity towards RBP4 that could be interfering with the ELISA measurement of RBP4. However, a previous study has shown that circulating RBP4 and TTR were not affected by human obesity or T2DM, compared to lean controls . The samIn conclusion, the present study documented RBP4 being a strong predictor of CAD, defined as angiographically significant coronary stenosis. That result was not influenced by acute (AMI) or stable (SA) CAD phase. Most importantly, RBP4 levels seemed to independently correlate to CAD severity. Thus, RBP4 could be a cost-effective, easy to obtain, novel risk biomarker, that could contribute to improved clinical decision making and management of patients at risk of CAD.