Towards an intraoperative engineering of osteogenic and vasculogenic grafts from the stromal vascular fraction of human adipose tissue.

Grafts generated by cultivation of progenitor cells from the stromal vascular fraction of human adipose tissue have been proven to have osteogenic and vasculogenic properties in vivo. However, in vitro manufacture of such implants is challenged by complex, impractical and expensive processes, and requires implantation in a separate surgery. This study investigates the feasibility of an intraoperative approach to engineer cell-based bone grafts with tissue harvest, cell isolation, cell seeding onto a scaffold and subsequent implantation within a few hours. Freshly isolated adipose tissue cells from a total of 11 donors, containing variable fractions of mesenchymal and endothelial progenitors, were embedded at different densities in a fibrin hydrogel, which was wrapped around bone substitute materials based on beta-tricalcium phosphate (ChronOS), hydroxyapatite (Engipore), or acellular xenograft (Bio-Oss). The resulting constructs, generated within 3 hours from biopsy harvest, were immediately implanted ectopically in nude mice and analysed after eight weeks. All explants contained blood vessels formed by human endothelial cells, functionally connected to the recipient's vasculature. Human origin cells were also found within osteoid structures, positively immunostained for bone sialoprotein and osteocalcin. However, even with the highest loaded cell densities, no frank bone tissue was detected, independently of the material used. These results provide a proof-of-principle that an intraoperative engineering of autologous cell-based vasculogenic bone substitutes is feasible, but highlight that - in the absence of in vitro commitment--additional cues (e.g., low dose of osteogenic factors or orthotopic environmental conditions) are likely needed to support complete osteoblastic cell differentiation and bone tissue generation.

Synthesis and biological activity of luteinizing hormone-releasing hormone and related peptides.

Syntheses of the decapeptide luteinizing hormone-releasing hormone, less thanGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 are described. The basic properties of arginine can provide a simple repetitive isolation procedure for arginine-containing peptides. The biological activities of the decapeptide, of a range of fragments and modified fragments, and of two analogs with alteration in the series at position 4 were measured by in vitro incubation with sheep pituitary slices, measuring the liberated LH by bioassay. None of the compounds of shortened sequence were active, with the exception of less thanGlu-His-Trp which showed 1% of the decapeptide in one of four experiments. Neither [Ser(But)4]-LH-RH-nor [Leu4]-LH-RH showed significant activity indicating (despite the known activity of [Ala4]-LH-RH) the importance of this part of the structure for full biological activity.

PIP: Numerous syntheses of luteinizing homrone-releasing hormone (LH-RH), with a decapeptide structure of less than (Glu-His-Ser-Tyr-Gly-Leu-Arg-P ro-Gly-NH2, are described. Arginine offers a simple repetitive isolation procedure for arginine-containing peptides. In vitro incubation with sheep pituitary slices and measurement of liberated LH by bioassay were utilized to examine the biological activities of the de capeptide, of a range of fragments and modified fragments, and of 2 analogs with alteration in the serine at position 4. Except for less than Glu-His-Trp, which showed 1% of the activity of the decapeptide in 1 of 4 experiments, none of these compounds of shortened sequence showed activity. Despite the established activity of Ala4-LH-RH, neither Ser (But) 4-LH-RH or Leu4-LH-RH showed significant activity.

[Metacarpal fractures: treatment indications and options. Results of a multicenter study]. / Frakturen der Mittelhand. Indikationen und Behandlungsoptionen.

This multicenter study analyzes data on metacarpal fractures in 1260 patients to determine optimal treatment concepts. Of the 740 patients followed up, 487 (66%) presented with fractures of a single metacarpal in one of the three most frequently observed locations (distal fractures of the 4th and 5th metacarpal, shaft fractures of the 2nd to 5th metacarpal, and fractures of the base of the first metacarpal). The overall clinical and radiological results were good to excellent. The "path" analysis used to identify baseline parameters and treatment options influencing outcome led to recommendations for metacarpal fracture treatment. Functional protocols are predominantly applied for postoperative/post-traumatic treatment. Pain and regional pain syndromes should be prevented whenever possible and early adequate treatment should be initiated due to their significant impact on outcome.

Detoxification of the plant toxin fluoroacetate by a genetically modified rumen bacterium.

We isolated the fluoroacetate dehalogenase gene (H1), from Moraxella species strain B, and placed it under the transcriptional control of a 154 bp fragment of the erm gene promoter. The promoter/gene construct was attached to the Butyrivibrio fibrisolvens shuttle vector pBHerm, and the resulting dehalogenase expression plasmid (pBHf) was transferred to B. fibrisolvens OB156 by electroporation. The erm gene promoter directed expression of dehalogenase activity in both E. coli and B. fibrisolvens OB156. Cell-free lysates of the genetically modified OB156 defluorinated 10.6 nmol fluoroacetate/min/mg protein. Growing cultures of OB156 were able to detoxify fluoroacetate in the culture medium, at the rate of 9.9 nmol/min/mg. Plasmid pBHf was retained by 100% of OB156 cells after 500 generations of non-selective culture. The restriction pattern of pBHf remained unchanged after extensive non-selective growth and host bacteria continued to produce active dehalogenase. The construction of rumen bacteria that are able to detoxify an important natural poison supports the feasibility of using genetically modified rumen bacteria to aid animal production.

[Perioperative coagulation management in microsurgery: report of the consensus workshops in the course of the 31st and 32nd Annual Meeting of the German-language Working Group for microsurgery of the peripheral nerves and vessels (DAM) November 2009 in Erlangen and November 2010 in Basel]. / Perioperatives Gerinnungsmanagement in der Mikrochirurgie - Bericht der Konsensus-Workshops im Rahmen der 31. und 32. Jahrestagung der Deutschsprachigen Arbeitsgemeinschaft für Mikrochirurgie der peripheren Nerven und Gefäße (DAM) im November 2009 in Erlangen und November 2010 in Basel.

Microsurgery is a very relevant component of reconstructive surgery. In this context anticoagulation plays an increasing role. At the moment there are no unanimously accepted prospective studies or generally accepted regimes available that could serve as evidence-based guidelines for the prevention of thrombosis in microsurgery. With regard to this problem the aim of a series of workshops during the annual meetings of the German-speaking group for microsurgery in 2009 and 2010 was to establish a first possible consensus. This article reflects the main aspects of the ongoing development of a generally acceptable guideline for anticoagulation in microsurgery as interim report of these consensus workshops. Basically there are 3 main agents in thromboprophylaxis available: antiplatelet drugs, dextran and heparin. In the course of the workshops no general use of aspirin or dextran for anticoagulation in microsurgery was recommended. The use of heparin as anticoagulation agent is advisable for different indications. Low molecular heparins (LMH) have certain advantages in comparison to unfractionated heparins (UFH) and are therefore preferred by most participants. Indications for UFH are still complex microsurgical revisions, renal failure and some specific constellations in patients undergoing reconstruction of the lower extremity, where the continuous administration of heparin is recommended. At the moment of clamp release a single-shot of UFH is still given by many microsurgeons, despite a lack of scientific evidence. Future prospective clinical trials and the establishment of a generally accepted evidence-based guideline regarding anticoagulation treatment in microsurgery are deemed necessary.

Comparison of short-term progestin-based protocols to synchronize estrus and ovulation in postpartum beef cows.

Two experiments evaluated short-term controlled internal drug release (CIDR) insert-based protocols to synchronize estrus and ovulation and compare differences in their potential to facilitate fixed-time AI (FTAI) in postpartum beef cows. Experiment 1 was designed to compare the 7- and 5-d Select Synch + CIDR protocols on the basis of timing and synchrony of estrus after treatment. Cows assigned to the 7-d protocol (n = 59) received GnRH [100 microg intramuscularly (i.m.)] and CIDR inserts (1.38 g of progesterone) on d 0 and PGF(2alpha) (25 mg i.m.) and CIDR removal on d 7. Cows assigned to the 5-d protocol (n = 58) received GnRH and CIDR inserts on d 2, PGF(2alpha) and CIDR removal on d 7, and a second injection of PGF(2alpha) 12 h after CIDR removal. Estrus detection and AI were performed for cows assigned to each protocol during the 144-h synchronized period. There was no difference in estrous response (P = 0.85), interval to estrus (P = 0.09), or variance for interval to estrus (P = 0.75) between treatments, nor were there differences in synchronized conception or pregnancy rates resulting from AI (P = 0.85, P = 0.91, respectively). Experiment 2 was designed to compare pregnancy rates resulting from FTAI after administration of the 7- and 5-d CO-Synch + CIDR protocols. Both treatments were administered the same as in Exp. 1; however, cows assigned to the 7-d protocol were inseminated 66 h after PGF(2alpha) and CIDR removal, and cows assigned to the 5-d protocol were inseminated 72 h after the first PGF(2alpha) injection. Cows assigned to both protocols were administered GnRH (100 microg i.m.) at AI. There was no effect of treatment (P = 0.85), technician (P = 0.20), or sire (P = 0.25) on pregnancy rates resulting from FTAI. Given these observations, the 5-d protocol provides an effective alternative to the 7-d protocol for use in facilitating FTAI; however, beef producers must carefully consider the increased labor and treatment costs associated with the 5-d protocol.

Effect of ovulatory follicle size and expression of estrus on progesterone secretion in beef cows.

Induced ovulation of small dominant follicles (SF, < 12 mm; CO-Synch protocol) in postpartum beef cows resulted in formation of corpora lutea (CL) that exhibited a delayed rise in progesterone (P4) compared with CL from large dominant follicles (LF, > 12 mm). Experiment 1 characterized P4 concentrations from ovulation to subsequent estrus among GnRH-induced or spontaneously ovulated SF (or= 12 mm) to determine if P4 secretion by CL formed from GnRH-induced SF remains lower postovulation in nonlactating beef cows. Nonlactating beef cows were induced to ovulate 48 h after PGF(2alpha) (CO-Synch; GnRH on d - 9, PGF(2alpha) on d - 2, and GnRH on d 0) or exhibited estrus and spontaneously ovulated after PGF(2alpha). Follicle size was measured at the second GnRH in cows induced to ovulate or approximately 3 h after the onset of estrus for cows that ovulated spontaneously. Cows were classified into 1 of 4 groups: 1) GnRH-induced ovulation-SF (or= 12 mm; Ind-LF; n = 16); 3) spontaneous ovulation-SF (or= 12 mm; Spon-LF; n = 22). Serum concentrations of P4 from d 3 to 15 were reduced in the Ind-SF compared with the Ind-LF (P = 0.05), Spon-SF (P = 0.07), and Spon-LF (P = 0.03). Experiment 2 characterized P4 concentrations (0 to 60 d postAI) among GnRH-induced or spontaneously ovulated SF (or= 13 mm) to determine if P4 secretion by CL formed from GnRH-induced SF remained lower during early gestation. Ovulation was induced with GnRH 48 h after PGF(2) (CO-Synch) or occurred spontaneously, and ovulatory follicle size was measured at AI. Lactating cows were classified into 1 of 3 groups: 1) GnRH-induced ovulation-SF (or= 13 mm; Ind-LF; n = 43); or 3) spontaneous ovulation-LF (>or= 13 mm; Spon-LF; n = 27). The increase in P4 concentrations was greater (P = 0.06) in pregnant (d 2 to 12) compared with nonpregnant cows. Also, the increase in P4 from d 2 to 12 was greater (P = 0.01) in the Ind-LF compared with the Ind-SF groups, but there was no difference (P = 0.94) among groups in P4 from d 14 to 60 in pregnant cows. Follicle size at AI influenced the increase in P4 in cows that failed to conceive (P = 0.007), but not among cows that became pregnant (P = 0.32) to AI. In summary, P4 secretion after GnRH-induced ovulation of SF was decreased from d 2 to 12 compared with that of LF, but was similar among pregnant cows from d 14 to 60 postAI (d 0).

Timing of artificial insemination in postpartum beef cows following administration of the CO-Synch + controlled internal drug-release protocol.

This experiment was designed to compare pregnancy rates in postpartum beef cows resulting from fixed-time AI (FTAI) at 54 or 66 h after administration of the CO-Synch + controlled internal drug-release (CIDR) protocol. Cows (n = 851) at 2 locations over 2 yr (yr 1, n = 218 and 206; and yr 2, n = 199 and 228 at the 2 locations, respectively) were stratified by age, BCS, and days postpartum to 1 of 2 FTAI intervals. Cows were administered GnRH (100 mug, i.m.) and were equipped with a CIDR insert (1.38 g of progesterone) on d 0. Controlled internal drug-release inserts were removed 7 d later at the time PGF(2alpha) (25 mg, i.m.) was administered (d 7). Continuous estrus detection was performed at location 2 by using the HeatWatch Estrus Detection System; the transmitters were fitted at the time of PGF(2alpha) and removed at the time of AI. Artificial insemination was performed at predetermined fixed times [54 h (FTAI 54; n = 424) or 66 h (FTAI 66; n = 427) after PGF(2alpha)] and all cows were administered GnRH (100 mug, i.m.) at AI. Two blood samples were collected on d -10 or -8 and immediately before treatment initiation to determine the pretreatment estrous cyclicity status of cows [progesterone >/=0.5 ng/mL (FTAI 54, 288/424 = 68%; FTAI 66, 312/427 = 73%; P = 0.07)]. Pregnancy rates were greater (P < 0.01) among cows that exhibited estrus than among those that did not (123/163 = 76% and 150/270 = 56%, respectively). There were no treatment x location interactions within year (P > 0.10) for age, days postpartum, or BCS; thus, the results were pooled for the respective treatments. Pregnancy rates were greater for FTAI 66 than FTAI 54 (P = 0.05; 286/426 = 67% vs. 257/424 = 61%, respectively). Pregnancy rates resulting from FTAI did not differ between year (P = 0.09), farm (P = 0.80), AI sire (P = 0.11), or technician (P = 0.64). There was no difference between pregnancy rates resulting from FTAI based on pretreatment cyclicity status (P = 0.30), and there was no difference between treatments in final pregnancy rates (P = 0.77). In summary, pregnancy rates resulting from FTAI following CO-Synch + CIDR at 66 h were greater than those resulting from FTAI at 54 h.

Comparison of progestin-based estrus synchronization protocols before fixed-time artificial insemination on pregnancy rate in beef heifers.

The objective of the experiment was to compare pregnancy rates resulting from fixed-time AI after administration of either 1 of 2 controlled internal drug release (CIDR)-based protocols. Heifers at 3 locations (location 1, n = 78; location 2, n = 61; and location 3, n = 78) were assigned to 1 of 2 treatments within reproductive tract scores (1 = immature to 5 = cycling) by age and BW. Heifers assigned to CIDR Select received a CIDR insert (1.38 g of progesterone) from d 0 to 14 followed by GnRH (100 mug, i.m.) 9 d after CIDR removal (d 23) and PGF2alpha (PG, 25 mg, i.m.) 7 d after GnRH treatment (d 30). Heifers assigned to CO-Synch + CIDR were administered GnRH and received a CIDR insert on d 23 and PG and CIDR removal on d 30. Heifers at location 1 were fitted with a HeatWatch estrus detection system transmitter from the time of PG until 24 d after fixed-time AI to allow for continuous estrus detection. Artificial insemination was performed at predetermined fixed times for heifers in both treatments at 72 or 54 h after PG for the CIDR Select and CO-Synch + CIDR groups, respectively. All heifers were administered GnRH at the time of AI. Blood samples were collected 10 d before and immediately before treatment initiation (d 0) to determine pretreatment estrous cyclicity (progesterone > or = 0.5 ng/mL). At location 1, the estrous response during the synchronized period was greater (P = 0.06; 87 vs. 69%, respectively), and the variance for interval to estrus after PG was reduced among CIDR Select- (P < 0.01) compared with CO-Synch + CIDR-treated heifers. Fixed-time AI pregnancy rates were significantly greater (P = 0.02) after the CIDR Select protocol (62%) compared with the CO-Synch + CIDR protocol (47%). In summary, the CIDR Select protocol resulted in a greater and more synchronous estrous response and significantly greater fixed-time AI pregnancy rates compared with the CO-Synch + CIDR protocol.

Comparison of progestin-based protocols to synchronize estrus and ovulation before fixed-time artificial insemination in postpartum beef cows.

This experiment was designed to compare pregnancy rates in postpartum beef cows resulting from fixed-time AI (FTAI) after treatment with 1 of 2 protocols to synchronize estrus and ovulation. Cross-bred, suckled beef cows (n = 650) at 4 locations (n = 210; n = 158; n = 88; and n = 194) were assigned within a location to 1 of 2 protocols within age group by days postpartum and BCS. Cows assigned to the melengestrol acetate (MGA) Select treatment (MGA Select; n = 327) were fed MGA (0.5 mg x head(-1) x d(-1)) for 14 d, GnRH (100 microg of Cystorelin i.m.) was injected on d 26, and prostaglandin F2alpha (PG; 25 mg of Lutalyse i.m.) was injected on d 33. Cows assigned to the CO-Synch + controlled internal drug release (CIDR) protocol (CO-Synch + CIDR; n = 323) were fed a carrier for 14 d, were injected with GnRH and equipped with an EAZI-BREED CIDR insert (1.38 g of progesterone, Pfizer Animal Health, New York, NY) 12 d after carrier removal, and PG (25 mg of Lutalyse i.m.) was injected and the CIDR were removed on d 33. Fixed-time AI was performed at 72 or 66 h after PG for the MGA Select or CO-Synch + CIDR groups, respectively. All cows were injected with GnRH (100 microg of Cystorelin i.m.) at the time of insemination. Blood samples were collected 8 and 1 d before the beginning of MGA or carrier to determine estrous cyclicity status of the cows (estrous cycling vs. anestrus) before treatment [progesterone > or = 0.5 ng/mL (MGA Select, 185/327, 57%; CO-Synch + CIDR, 177/323, 55%); P = 0.65]. There was no difference (P = 0.20) in pregnancy rate to FTAI between treatments (MGA Select, 201/327, 61%; CO-Synch + CIDR, 214/323, 66%). There was also no difference (P = 0.25) between treatments in final pregnancy rate at the end of the breeding period (MGA Select, 305/327, 93%; CO-Synch + CIDR, 308/323, 95%). These data indicate that pregnancy rates to FTAI were comparable after administration of the MGA Select or CO-Synch + CIDR protocols. Both protocols provide opportunities for beef producers to utilize AI and potentially eliminate the need to detect estrus.

A comparison of progestin-based protocols to synchronize ovulation and facilitate fixed-time artificial insemination in postpartum beef cows.

The experimental objective was to compare pregnancy rates after fixed-time AI in postpartum suckled beef cows following administration of two progestin-based protocols to synchronize ovulation. Cows (n = 424) at three locations (n = 208, 122, and 92 per location) were stratified by age, BCS, and days postpartum (DPP) and assigned randomly to one of the two treatment protocols. The MGA Select-treated cows (MGA Select; n = 213) were fed melengestrol acetate (MGA, 0.5 mg x cow(-1) x d(-1)) for 14 d and carrier for 8 d, and then GnRH (100 microg i.m. Cystorelin; d 26) was injected 12 d after MGA withdrawal, and PG (25 mg i.m. Lutalyse) was administered 7 d after GnRH. Cows assigned to the 7-11 Synch protocol (7-11 Synch; n = 209) were fed carrier for 15 d and MGA for 7 d, and then injected with PG on d 22 (d 7 of MGA), GnRH on d 26, and PG again on d 33. Artificial insemination was performed at fixed times for cows in both treatments at 60 or 72 h after d 33 PG for 7-11 Synch and MGA Select groups, respectively. All cows were injected with GnRH (100 microg of i.m. Cystorelin) at AI. There was no treatment x location interaction for age (P = 0.90), BCS (P = 0.64), or DPP (P = 0.93), and the results were therefore pooled for the respective treatments (age [7-11 Synch, 5.5 +/- 0.2; MGA Select, 5.5 +/- 0.2], BCS [7-11 Synch, 5.7 +/- 0.1; MGA Select, 5.6 +/- 0.1], and DPP [7-11 Synch, 41.1 +/- 1.1; MGA Select, 42.1 +/- 1.1]). Blood samples were collected 8 and 1 d before MGA or carrier to determine pretreatment estrous cyclicity (progesterone >or=1 ng/mL; 7-11 Synch, 59/209 [28%]; MGA Select, 54/213 [25%]; P = 0.50) and again on d 33 PG to evaluate treatment response as a percentage of cows with progesterone concentrations in serum >or=1ng/mL (7-11 Synch, 184/209 [88%]; MGA Select, 177/213 [83%]; P = 0.15). Pregnancy rates resulting from fixed-time AI did not differ (P = 0.25) between treatments (7-11 Synch, 128/209 [61%]; MGA Select, 142/213 [67%]), nor did pregnancy rates (P = 0.77) at the end of the breeding season (7-11 Synch, 198/208 [95%]; MGA Select, 204/213 [96%]). These data indicate that pregnancy rates were comparable after fixed-time AI, following administration of the 7-11 Synch and MGA Select protocols. Both protocols provide opportunities for beef producers to use AI and eliminate the need to detect estrus.

Genetics and linkage mapping of Drosophila buzzatii.

Of 51 visible mutants isolated from natural or laboratory populations of Drosophila buzzatii, or X-ray induced, 42 have been assigned to chromosomes, and linkage maps have been constructed. About half of the autosomal mutants map to chromosome 2, with only two on chromosome 3 and none on chromosome 4. For the whole repleta group, chromosome 2 also exhibits much greater inversion variability than other chromosomes, which suggests variation among chromosomes in apparent mutability. The chromosomes of D. buzzatii are homologized to those of D. melanogaster and to the standard chromosomal elements of Drosophila. Sequence comparisons for six X chromosome mutant genes, whose homology is reasonably certain, in 13 Drosophila species confirm linkage group conservation but great variation among species in gene order. The linkage group conservation of single-copy genes stands in contrast to observed transpositions between elements for tandem repeat genes.