Comparison of reproductive management programs for submission of Holstein heifers for first insemination with conventional or sexed semen based on expression of estrus, pregnancy outcomes, and cost per pregnancy.

Our objective was to evaluate reproductive management programs for submission of Holstein heifers for first insemination with conventional or sexed semen. In experiment 1, nulliparous Holstein heifers (n = 462) were submitted to a 5-d progesterone-releasing intravaginal device (PRID)-Synch protocol [d 0, GnRH + PRID; d 5, PGF2α - PRID; d 6, PGF2α; d 8, GnRH + TAI] and were randomly assigned for PRID removal on d 5 or 6 of the protocol followed by timed artificial insemination (TAI) with conventional semen. Delaying PRID removal decreased early expression of estrus before scheduled TAI (0.9 vs. 12.2%), and pregnancies per AI (P/AI) did not differ between treatments. In experiment 2, nulliparous Holstein heifers (n = 736) from 3 commercial farms were randomized within farm to 1 of 3 treatments for first AI with sexed semen: (1) CIDR5 [d -6, GnRH + controlled internal drug release (CIDR); d -1, PGF2α - CIDR; d 0, PGF2α; d 2, GnRH + TAI]; (2) CIDR6 (d -6, GnRH + CIDR; d -1, PGF2α; d 0, PGF2α - CIDR; d 2, GnRH + TAI); and (3) EDAI (PGF2α on d 0 followed by once-daily estrous detection and AI). Delaying CIDR removal decreased early expression of estrus before scheduled TAI (0.004 vs. 27.8%); however, CIDR5 heifers tended to have more P/AI at 35 (53 vs. 45 vs. 46%) and 64 (52 vs. 45 vs. 45%) days after AI than CIDR6 and EDAI heifers, respectively. Overall, CIDR5 and CIDR6 heifers had fewer days to first AI and pregnancy than EDAI heifers which resulted in less feed costs than EDAI heifers due to fewer days on feed until pregnancy. Despite greater hormonal treatment costs for CIDR5 heifers, costs per pregnancy were $16.66 less for CIDR5 than for EDAI heifers. In conclusion, delaying PRID removal by 24 h within a 5-d PRID-Synch protocol in experiment 1 suppressed early expression of estrus before TAI, and P/AI for heifers inseminated with conventional semen did not differ between treatments. By contrast, although delaying CIDR removal by 24 h within a 5-CIDR-Synch protocol in experiment 2 suppressed early expression of estrus before TAI, delaying CIDR removal by 24 h tended to decrease P/AI for heifers inseminated with sexed semen. Further, submission of heifers to a 5-d CIDR-Synch protocol for first AI tended to increase P/AI and decrease the cost per pregnancy compared with EDAI heifers.

Short communication: Effect of timing of induction of ovulation relative to timed artificial insemination using sexed semen on pregnancy outcomes in primiparous Holstein cows.

Our objective was to determine the effect of increasing the interval from induction of ovulation to timed artificial insemination (TAI) on fertility by decreasing the interval from TAI to ovulation using sexed semen within a synchronized breeding program. Our hypothesis was that induction of ovulation earlier relative to TAI would increase pregnancies per artificial insemination (P/AI). Primiparous Holstein cows from 3 commercial dairy farms in the United States were submitted to a Double-Ovsynch protocol for first service as follows: Pre-Ovsynch (GnRH; 7 d, PGF2α; 3 d, GnRH), followed 7 d later by Breeding-Ovsynch [GnRH (G1); 7 d, PGF2α; 24 h, PGF2α], followed by the last GnRH treatment (G2), which varied between treatments, and TAI. To vary the interval between G2 and TAI, cows were randomized to 2 treatments to receive G2 either 16 (G2-16; n = 373) or 24 (G2-24; n = 357) h before TAI, which was fixed at 48 h after the second PGF2α treatment of the Breeding-Ovsynch portion of the protocol. All cows were inseminated with sexed semen, and each herd used sires of their choosing, which were randomly allocated between treatments. Pregnancy diagnosis was conducted by herd veterinarians using transrectal ultrasonography. In disagreement with our hypothesis, G2-24 cows had fewer P/AI than G2-16 cows at 34 ± 3 d (44 vs. 50%) and 80 ± 17 d (41 vs. 48%) after TAI. Pregnancy loss (5 vs. 6%) and fetal sex ratio (92:8 vs. 90:10, female:male) did not differ between treatments for G2-16 and G2-24 cows, respectively. Thus, we reject our hypothesis and conclude that induction of ovulation earlier relative to TAI with sexed semen for first service after a Double-Ovsynch protocol decreased P/AI in primiparous Holstein cows.

A 96-well microplate bioreactor platform supporting individual dual perfusion and high-throughput assessment of simple or biofabricated 3D tissue models.

Traditional 2D monolayer cell cultures and submillimeter 3D tissue construct cultures used widely in tissue engineering are limited in their ability to extrapolate experimental data to predict in vivo responses due to their simplistic organization and lack of stimuli. The rise of biofabrication and bioreactor technologies has sought to address this through the development of techniques to spatially organize components of a tissue construct, and devices to supply these tissue constructs with an increasingly in vivo-like environment. Current bioreactors supporting both parenchymal and barrier tissue constructs in interconnected systems for body-on-a-chip platforms have chosen to emphasize study throughput or system/tissue complexity. Here, we report a platform to address this disparity in throughput and both system complexity (by supporting multiple in situ assessment methods) and tissue complexity (by adopting a construct-agnostic format). We introduce an ANSI/SLAS-compliant microplate and docking station fabricated via stereolithography (SLA), or precision machining, to provide up to 96 samples (Ø6 × 10 mm) with two individually-addressable fluid circuits (192 total), loading access, and inspection window for imaging during perfusion. Biofabricated ovarian cancer models were developed to demonstrate the in situ assessment capabilities via microscopy and a perfused resazurin-based metabolic activity assay. In situ microscopy highlighted flexibility of the sample housing to accommodate a range of sample geometries. Utility for drug screening was demonstrated by exposing the ovarian cancer models to an anticancer drug (doxorubicin) and generating the dose-response curve in situ, while achieving an assay quality similar to static wellplate culture. The potential for quantitative analysis of temporal tissue development and screening studies was confirmed by imaging soft- (gelatin) and hard-tissue (calcium chloride) analogs inside the bioreactor via spectral computed tomography (CT) scanning. As a proof-of-concept for particle tracing studies, flowing microparticles were visualized to inform the design of hydrogel constructs. Finally, the ability for mechanistic yet high-throughput screening was demonstrated in a vascular coculture model adopting endothelial and mesenchymal stem cells (HUVEC-MSC), encapsulated in gelatin-norbornene (gel-NOR) hydrogel cast into SLA-printed well inserts. This study illustrates the potential of a scalable dual perfusion bioreactor platform for parenchymal and barrier tissue constructs to support a broad range of multi-organ-on-a-chip applications.

The histone demethylase KDM3A, and its downstream target MCAM, promote Ewing Sarcoma cell migration and metastasis.

Ewing Sarcoma is the second most common solid pediatric malignant neoplasm of bone and soft tissue. Driven by EWS/Ets, or rarely variant, oncogenic fusions, Ewing Sarcoma is a biologically and clinically aggressive disease with a high propensity for metastasis. However, the mechanisms underpinning Ewing Sarcoma metastasis are currently not well understood. In the present study, we identify and characterize a novel metastasis-promotional pathway in Ewing Sarcoma, involving the histone demethylase KDM3A, previously identified by our laboratory as a new cancer-promoting gene in this disease. Using global gene expression profiling, we show that KDM3A positively regulates genes and pathways implicated in cell migration and metastasis, and demonstrate, using functional assays, that KDM3A promotes migration in vitro and experimental, post-intravasation, metastasis in vivo. We further identify the melanoma cell adhesion molecule (MCAM) as a novel KDM3A target gene in Ewing Sarcoma, and an important effector of KDM3A pro-metastatic action. Specifically, we demonstrate that MCAM depletion, like KDM3A depletion, inhibits cell migration in vitro and experimental metastasis in vivo, and that MCAM partially rescues impaired migration due to KDM3A knock-down. Mechanistically, we show that KDM3A regulates MCAM expression both through a direct mechanism, involving modulation of H3K9 methylation at the MCAM promoter, and an indirect mechanism, via the Ets1 transcription factor. Finally, we identify an association between high MCAM levels in patient tumors and poor survival, in two different Ewing Sarcoma clinical cohorts. Taken together, our studies uncover a new metastasis-promoting pathway in Ewing Sarcoma, with therapeutically targetable components.

Serum, uterine, and vaginal mucosal IgG antibody responses against Tritrichomonas foetus after administration of a commercial killed whole T foetus vaccine in beef cows.

The objective of this study was to determine the level and duration of IgG antibodies induced against killed whole Tritrichomonas foetus and T foetus-purified surface antigen (TF1.17) in serum, vaginal, and uterine secretions after systemic immunization of beef cows with a vaccine containing killed whole T foetus. Twenty nonpregnant beef cows were randomly assigned to vaccine or control groups as follows: Vaccine (n = 10): cows received 2 mL of a commercial vaccine containing killed whole T foetus subcutaneously and a 2-mL booster 2 weeks later. Control (n = 10): cows received 2 mL of sterile saline on the same schedule. Vaginal secretions and blood samples were collected on Days 0, 8, 15, 22, 29, 36, 43, 50, 60, 75, 89, 110, 146, and 182 relative to day of primary vaccination. Uterine flush fluid was collected on Days 0, 15, 29, and 43 after the day of primary vaccination. Samples were assayed for IgG antibodies to the killed whole T foetus and surface antigen TF1.17 using enzyme-linked immunosorbent assay. Serum whole T foetus-specific IgG levels were significantly increased (between Days 15 and 182) following vaccination with T foetus or with saline. No differences between vaccinates and controls in uterine responses to whole-cell antigen were detected. Serum anti-TF1.17 IgG responses to vaccination were significantly higher than Day 0 throughout the immunization period (P < 0.001) and were higher than responses in control animals on each day post immunization through Day 146 (P < 0.001). A significant rise in TF1.17-specific IgG levels was observed in vaginal and uterine fluids from Day 15 post vaccination compared to the Day 0 levels. These levels remained significantly elevated in vaginal and uterine fluids through Days 75 (P < 0.05) and 43 (P < 0.001) after primary vaccination, respectively. Antibody levels in serum, vaginal, and uterine secretions against TF1.17 remained low in the control group throughout the study. In conclusion, vaccination of beef cows with a commercial vaccine containing T foetus induced significant increase in the levels of IgG to the T foetus TF1.17 surface antigen in serum, vaginal secretions, and uterine fluid, which remained elevated through Days 43, 75, and 182 in uterine fluids, vaginal secretions, and serum, respectively. Since purified TF1.17 antigen has been shown to protect against experimental T foetus infection in heifers, the vaccine-induced TF1.17-specific IgG response is likely to be important in the prevention of trichomoniasis in beef cattle.

The histone demethylase KDM3A is a microRNA-22-regulated tumor promoter in Ewing Sarcoma.

Ewing Sarcoma is a biologically aggressive bone and soft tissue malignancy affecting children and young adults. Ewing Sarcoma pathogenesis is driven by EWS/Ets fusion oncoproteins, of which EWS/Fli1 is the most common. We have previously shown that microRNAs (miRs) regulated by EWS/Fli1 contribute to the pro-oncogenic program in Ewing Sarcoma. Here we show that miR-22, an EWS/Fli1-repressed miR, is inhibitory to Ewing Sarcoma clonogenic and anchorage-independent cell growth, even at modest overexpression levels. Our studies further identify the H3K9me1/2 histone demethylase KDM3A (JMJD1A/JHDM2A) as a new miR-22-regulated gene. We show that KDM3A is overexpressed in Ewing Sarcoma, and that its depletion inhibits clonogenic and anchorage-independent growth in multiple patient-derived cell lines, and tumorigenesis in a xenograft model. KDM3A depletion further results in augmentation of the levels of the repressive H3K9me2 histone mark, and downregulation of pro-oncogenic factors in Ewing Sarcoma. Together, our studies identify the histone demethylase KDM3A as a new, miR-regulated, tumor promoter in Ewing Sarcoma.

A novel oncogenic mechanism in Ewing sarcoma involving IGF pathway targeting by EWS/Fli1-regulated microRNAs.

MicroRNAs (miRs) are a novel class of cellular bioactive molecules with critical functions in the regulation of gene expression in normal biology and disease. MiRs are frequently misexpressed in cancer, with potent biological consequences. However, relatively little is known about miRs in pediatric cancers, including sarcomas. Moreover, the mechanisms behind aberrant miR expression in cancer are poorly understood. Ewing sarcoma is an aggressive pediatric malignancy driven by EWS/Ets fusion oncoproteins, which are gain-of-function transcriptional regulators. We employed stable silencing of EWS/Fli1, the most common of the oncogenic fusions, and global miR profiling to identify EWS/Fli1-regulated miRs with oncogenesis-modifying roles in Ewing sarcoma. In this report, we characterize a group of miRs (100, 125b, 22, 221/222, 27a and 29a) strongly repressed by EWS/Fli1. Strikingly, all of these miRs have predicted targets in the insulin-like growth factor (IGF) signaling pathway, a pivotal driver of Ewing sarcoma oncogenesis. We demonstrate that miRs in this group negatively regulate the expression of multiple pro-oncogenic components of the IGF pathway, namely IGF-1, IGF-1 receptor, mammalian/mechanistic target of rapamycin and ribosomal protein S6 kinase A1. Consistent with tumor-suppressive functions, these miRs manifest growth inhibitory properties in Ewing sarcoma cells. Our studies thus uncover a novel oncogenic mechanism in Ewing sarcoma, involving post-transcriptional derepression of IGF signaling by the EWS/Fli1 fusion oncoprotein via miRs. This novel pathway may be amenable to innovative therapeutic targeting in Ewing sarcoma and other malignancies with activated IGF signaling.

Positive effect of FSH but not LH on early development of the dominant follicle in mares.

The effects of FSH, LH or both on follicular growth and intrafollicular free insulin-like growth factor (IGF)-1 and oestradiol were investigated in mares after the beginning of deviation (largest follicle >/= 20 mm; Hour 0). A single treatment with a gonadotropin-releasing hormone antagonist (acyline) was given at Hour 3 to suppress the concentrations of FSH and LH. Five groups (n = 5 mares per group) were evaluated in the present study: (1) control; (2) acyline treated; (3) acyline + recombinant equine (re) FSH treated; (4) acyline + reLH treated; and (5) combined acyline + reFSH + reLH treated. Beginning at Hour 3, reFSH and reLH were given at 6-h intervals in eight decreasing or increasing doses, respectively. The reFSH and reLH prevented the acyline-induced decreases in FSH and LH, respectively. Diameters and concentrations of intrafollicular free IGF-1 and oestradiol of the two largest follicles at Hour 48 did not differ significantly between the control and acyline + FSH groups, but were reduced (P < 0.05) similarly in the acyline and acyline + LH groups. The combination of reFSH and reLH was no more effective than reFSH alone. The results demonstrate a role for FSH but not LH in the growth of the largest follicle and intrafollicular concentrations of free IGF-1 and oestradiol during the 48 h after the beginning of deviation in mares.

Functional analysis of the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) enzyme, involved in the late steps of coenzyme B12 biosynthesis in Salmonella enterica.

In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT(WT)) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT(WT). The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.

Functional relationships among intrafollicular insulin-like growth factor 1, circulatory gonadotropins, and development of the dominant follicle in mares.

The functional relationships among intrafollicular free insulin-like growth factor 1 (IGF1), circulatory gonadotropins, and development of the dominant follicle were studied in 40 mares in two experiments. A GnRH antagonist (Acyline) was given i.m. at the expected beginning of follicular deviation (largest follicle or F1> or =20mm; Day 0) alone (Acyline group) or in combination with intrafollicular treatment of F1 with rhIGF1 (Acyline/IGF1 group). In Experiment 1, blood samples, follicular-fluid samples, and diameter of F1 were taken on Days 1 and 2. In Experiment 2, daily follicular diameter and blood samples were taken from Day 0 to ovulation. The GnRH antagonist induced a 50% decrease in circulatory FSH concentrations for 1 d and in LH for 2 d. In Experiment 1, control and Acyline/IGF1 groups had greater intrafollicular free IGF1 (P<0.05) and inhibin-A concentrations (P<0.08) than the Acyline group. The intrafollicular concentration of estradiol on Day 2 was greater (P<0.05) in the control group than in the Acyline and the Acyline/IGF1 groups. In Experiment 2, a decrease in diameter of F1 in the Acyline group was followed by a new follicular wave. All IGF-treated follicles grew and ovulated. Results indicated that the increase in intrafollicular free IGF1 observed in F1 in association with deviation is gonadotropin dependent. During the period of lesser gonadotropin concentrations from Acyline treatment, intrafollicular IGF1 stimulated follicular growth and inhibin concentrations, but not intrafollicular estradiol production.

Effect of suppression of FSH with a GnRH antagonist (acyline) before and during follicle deviation in the mare.

A GnRH antagonist (Acyline) was used to study the role of FSH in early development of a follicular wave in 61 mares. In Experiment 1, a single dose of 3 mg per mare, compared with 0 and 1 mg, suppressed both the FSH and follicle responses to exogenous GnRH. In Experiment 2, high concentrations of FSH were induced by two successive ablations of all follicles >/= 6 mm on days 10 and 13 (day 0 = ovulation). A single treatment with Acyline resulted in significantly greater suppression of plasma concentrations of FSH than a single treatment with charcoal-extracted follicular fluid (source of inhibin) or oestradiol. Suppression of FSH was not significantly different between the group treated with Acyline alone and a group treated with a combination of Acyline, inhibin and oestradiol. In Experiment 3, all follicles were ablated on day 10 to induce an FSH surge and a new follicular wave. Acyline treatment on day 10 resulted in an immediate decrease in FSH, without a significant effect on day of emergence of a new wave or growth of follicles from 7 to 11 mm on days 11-13. Treatment on day 15, a day before expected follicle deviation and after the peak of the wave-stimulating FSH surge, resulted in an immediate decrease in FSH and cessation of follicle growth. Results indicated that growth of follicles for about 2 days after wave emergence was independent of FSH. In contrast, during the decline in the wave-stimulating FSH surge and before follicle deviation, growth of follicles was dependent on FSH.

Dynamics of global transcriptome in bovine matured oocytes and preimplantation embryos.

Global activation of the embryonic genome is the most critical event in early mammalian development. After fertilization, a rich supply of maternal proteins and RNAs support development whereas a number of zygotic and embryonic genes are expressed in a stage-specific manner leading to embryonic genome activation (EGA). However, the identities of embryonic genes expressed and the mechanism(s) of EGA are poorly defined in the bovine. Using the Affymetrix bovine-specific DNA microarray as the biggest available array at present, we analyzed gene expression at two key stages of bovine development, matured oocytes (MII) and 8-cell-stage embryos, constituting the ultimate reservoir for life and a stage during which EGA takes place, respectively. Key genes in regulation of transcription, chromatin-structure cell adhesion, and signal transduction were up-regulated at the 8-cell stage as compared with 8-cell embryos treated with alpha-amanitin and MII. Genes controlling DNA methylation and metabolism were up-regulated in MII. These changes in gene expression, related to transcriptional machinery, chromatin structure, and the other cellular functions occurring during several cleavage stages, are expected to result in a unique chromatin structure capable of maintaining totipotency during embryogenesis and leading to differentiation during postimplantation development. Dramatic reprogramming of gene expression at the onset of development also has implications for cell plasticity in somatic cell nuclear transfer, genomic imprinting, and cancer.

Coccidioidomycosis: a descriptive survey of a reemerging disease. Clinical characteristics and current controversies.

Coccidioidomycosis is a fungal disease with protean manifestations endemic to the Lower Sonoran Life Zone, which includes the hot deserts of the southwestern United States and areas of Mexico. Two hundred and twenty-three patients were found to have coccidioidomycosis at our institution from 1994-2002, the largest reported cohort of coccidioidomycosis patients since the 1950s. Of these patients, 58% presented with isolated pulmonary disease, 14% had high (>1:16) complement fixation titers without clear evidence of dissemination, 22% had definite disseminated disease, and 5% had unclassified disease. Enzyme immunoassay was a reliable diagnostic tool in those with symptomatic disease, but had a low specificity in those who were asymptomatic. Complement fixation titers of > or =1:16 were associated with dissemination to bone or skin but were not helpful in evaluating central nervous system disease. Thirteen percent of patients with high complement fixation titers (>1:16) without clear evidence of dissemination on presentation and 7% of those with isolated pulmonary disease eventually progressed to disseminated disease; 30% of Filipino patients with pulmonary disease progressed to disseminated disease. Nonwhite race was a predictor for dissemination; African American patients more often developed disseminated bony disease while Filipinos were more likely to develop cutaneous or central nervous system disease. Relapse of disseminated coccidioidomycosis occurred in 24% of patients; the risk was highest (71%) among those with central nervous system disease. Azole therapy was generally inferior to amphotericin B in disseminated disease. Predictors of permanent disability included African American or Filipino race, central nervous system disease, and bony disease.

Mitochondrial endonuclease G is important for apoptosis in C. elegans.

Programmed cell death (apoptosis) is a tightly regulated process of cell disassembly in which dying cells and their nuclei shrink and fragment and the chromosomal DNA is degraded into internucleosomal repeats. Here we report the characterization of the cps-6 gene, which appears to function downstream of, or in parallel to, the cell-death protease CED-3 of Caenorhabditis elegans in the DNA degradation process during apoptosis. cps-6 encodes a homologue of human mitochondrial endonuclease G, and its protein product similarly localizes to mitochondria in C. elegans. Reduction of cps-6 activity caused by a genetic mutation or RNA-mediated interference (RNAi) affects normal DNA degradation, as revealed by increased staining in a TUNEL assay, and results in delayed appearance of cell corpses during development in C. elegans. This observation provides in vivo evidence that the DNA degradation process is important for proper progression of apoptosis. CPS-6 is the first mitochondrial protein identified to be involved in programmed cell death in C. elegans, underscoring the conserved and important role of mitochondria in the execution of apoptosis.

(E)-1-(2'-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-(2-iodovinyl)benzene.

This analysis of the title compound, C13H13F2IO3, establishes the orientation of (E)-5-(CH=CH-I) as antiperiplanar (ap) to the C-C bond (5-6 position) of the 2,4-difluorophenyl ring system, with the (E)-5-(CH=CH-I) H atom located in close proximity (2.17 A) to the F4 atom of the 2,4-difluorophenyl moiety.

Demonstration of the in vivo interaction of key cell death regulators by structure-based design of second-site suppressors.

Demonstrating in vivo interaction of two important biomolecules and the relevance of the interaction to a biological process have been difficult issues in biomedical research. Here, we report the use of a homology modeling approach to establish the significance of protein interactions in governing the activation of programmed cell death in Caenorhabditis elegans. A protein interaction cascade has been postulated to mediate activation of cell death in nematodes, in which the BH3-domain-containing (Bcl-2 homology region 3) protein EGL-1 binds the cell-death inhibitor CED-9 and induces release of the death-activating protein CED-4 from inhibitory CED-4/CED-9 complexes. We show here that an unusual gain-of-function mutation in ced-9 (substitution of glycine 169 to glutamate) that results in potent inhibition of most nematode cell deaths impairs the binding of EGL-1 to CED-9 and EGL-1-induced release of CED-4 from CED-4/CED-9 complexes. Based on a modeled EGL-1/CED-9 complex structure, we generated second-site compensatory mutations in EGL-1 that partially restore the binding of EGL-1 to CED-9(G169E) and EGL-1-induced release of CED-4 from CED-4/CED-9(G169E) complexes. Importantly, these mutations also significantly suppress the death-protective activity of CED-9(G169E) in vivo. These results establish that direct physical interaction between EGL-1 and CED-9 is essential for the release of CED-4 and the activation of cell death. The structure-based design of second-site suppressors via homology modeling should be widely applicable for probing important molecular interactions that are implicated in fundamental biological processes.

Laser ablation of burns: initial clinical trial.

BACKGROUND: Scanning, high-powered carbon dioxide laser ablation of eschar may facilitate blood conservation in patients with burns. METHODS: Twenty-one children with full-thickness burns that required serial excisions were enrolled in a Human Studies Committee approved protocol in which a full-thickness wound was ablated with a rapidly scanned continuous wave carbon dioxide laser system. A control wound was sharply excised, and both wounds were immediately autografted. End points were engraftment at 7 days and serial Vancouver scar scores. RESULTS: The children had an average age of 8.3 +/- 1.2 years, weight of 36.3 +/- 4.9 kg, and burn size of 40% +/- 5.1%. The study wounds were ablated with an average energy of 99.2 +/- 5.7 W; there was no bleeding from 19 successfully ablated wounds. Initial engraftment averaged 94.7% +/- 3.5% in the control sites and 94.7% +/- 3.3% in the study sites (P = 1.0). There was no significant difference in Vancouver scar scores at an average follow-up of 32.0 +/- 5.2 weeks. CONCLUSIONS: This pilot study follows a successful trial of this concept in a porcine model and demonstrates the technical feasibility of laser vaporization of burn eschar in humans with immediate autografting. Further refinement of the technique is required before it can be generally recommended.