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Background Robotic hepatectomy (RH) is increasingly utilized for minor and major liver resections. The IWATE criteria were developed to classify minimally invasive liver resections by difficulty. The objective of this study was to apply the IWATE criteria in RH and to describe perioperative and oncologic outcomes of RH over the last decade at our institution. Methods Perioperative and oncologic outcomes of patients who underwent RH between 2011 and 2019 were retrospectively collected. The difficulty level of each operation was assessed using the IWATE criteria, and outcomes were compared at each level. Univariate linear regression was performed to characterize the relationship between IWATE criteria and perioperative outcomes (OR time, EBL, and LOS), and a multivariable model was also developed to address potential confounding by patient characteristics (age, sex, BMI, prior abdominal surgery, ASA class, and simultaneous non-hepatectomy operation). Results Two hundred and twenty-five RH were performed. Median IWATE criteria for RH were 6 (IQR 5–9), with low, intermediate, advanced, and expert resections accounting for 23% (n = 51), 34% (n = 77), 32% (n = 72), and 11% (n = 25) of resections, respectively. The majority of resections were parenchymal-sparing approaches, including anatomic segmentectomies and non-anatomic partial resections. 30-day complication rate was 14%, conversion to open surgery occurred in 9 patients (4%), and there were no deaths within 30 days postoperatively. In the univariate linear regression analysis, IWATE criteria were positively associated with OR time, EBL, and LOS. In the multivariable model, IWATE criteria were independently associated with greater OR time, EBL, and LOS. Two-year overall survival for hepatocellular carcinoma and intrahepatic cholangiocarcinoma was 94% and 50%, respectively. Conclusion In conclusion, the IWATE criteria are associated with surgical outcomes after RH. This series highlights the utility of RH for difficult hepatic resections, particularly parenchymal-sparing resections in the posterosuperior sector, extending the indication of minimally invasive hepatectomy in experienced hands and potentially offering select patients an alternative to open hepatectomy or other less definitive liver-directed treatment options.

Prolonged static stretching (SS) in isolation (no dynamic warm-up) can impair muscle performance. There are conflicting reports whether impairments are present in antagonist and contralateral muscles. The objective of this study was to assess the effect of unilateral hamstrings SS on ipsilateral stretched and contralateral limbs’ strength and jump power. The SS (four repetitions of 30-s) and control sessions involved unilateral testing of the stretched leg and contralateral leg for knee extension (KE) maximum voluntary isometric contraction (MVIC) force and electromyography (EMG), drop jump (DJ) height and contact time at 1-min post-stretching. There were significant KE MVIC force impairments for both the SS (p = 0.006, d = 0.3, − 8.1%) and contralateral (p = 0.02, d = 0.20, − 4.2%) leg. With normalized data, there was a near-significant (p = 0.1), small magnitude (d = 0.29), greater force impairment with the ipsilateral (93.0 ± 12.8% of pre-test) versus the contralateral (96.2 ± 9.1% of pre-test) KE MVIC force. DJ height significantly improved for the stretched leg (p = 0.03, d = 0.18, + 9.2%) with near-significant, improvements for the contralateral leg (p = 0.06, d = 0.22, + 12.1%). For the stretched leg, DJ contact time was significantly (p = 0.04, d = 0.18, + 3.4%) prolonged, but there was no significant change with the contralateral leg. Unilateral hamstrings SS induced strength deficits in the ipsilateral and contralateral knee extension MVIC and a prolongation of the stretched leg DJ contact period. In anticipation of maximal force outputs, prolonged SS in isolation (no dynamic warm-up included) can have negative consequences on antagonist and contralateral muscle performance.

In this paper, the procedure of developing and evaluating a UAV-borne mapping system is described. The system is equipped with both a LiDAR and a camera. The system mounting parameters, as well as the intrinsic parameters of the individual sensors, are calibrated rigorously. Simultaneous calibration of the LiDAR intrinsic parameters and the LiDAR-camera mounting parameters is performed in a self-calibrating bundle adjustment with additional relative orientation constraints. A visual-inertial approach is proposed to georeference the laser scans without using a GNSS receiver. This approach is motivated not only by the interest of users in low-cost systems but also by the fact that the integrity of GNSS signals might be affected under several environmental conditions, e.g., indoors, in urban canyons, under tree canopies. It is shown that a low-cost inertial measurement unit not equipped with a dual-frequency, real-time kinematic GNSS receiver is still useful for georeferencing the laser scanning data with cm-level accuracy. The scans are also textured using the images captured by the camera, which enriches the LiDAR point clouds with spectral information.

Some of the structures affected by ASR are bridges with thick concrete slabs without shear reinforcement. This implies that concrete is the only component resisting to shear forces, which might be a problem considering that ASR can progressively and significantly affect the mechanical properties of that material. A study was launched to gain a better understanding of the effects of ASR on the behaviour of thick concrete slabs without shear reinforcement. The methodology developed for this study, as well as the analyses of the deformations/expansions obtained from different surface measurements, are presented in this paper. It led to interesting observations about the heterogeneity of the deformations between slabs. The expansion was monitored extensively in the longitudinal, vertical and transversal orientation. The expansion was the greatest in the transversal orientation reaching up to 0.6–0.7%. The steel reinforcement at the base of the specimens locally limited the expansion of the slabs and as such created an expansion gradient which increased with the advancement of the alkali-silica reaction. Also, the use of lithium nitrate proved successful as a mitigation method against the development of expansion due to ASR.

Angiotensin 1-7 (ANG-(1-7)), a derivative of angiotensin I or II, is involved in the propagation of sympathetic output to the heart and vasculature, and the receptor for ANG-(1-7), the Mas receptor, is expressed on astrocytes in the rostral ventrolateral medulla (RVLM). We recorded blood pressure (BP) and splanchnic sympathetic nerve activity (SSNA) before and after focal injection of ANG-(1-7) into the RVLM of rats. Unilateral injection of ANG-(1-7) into the RVLM, acting through the Mas receptor, increased SSNA and BP, and glutamate receptor antagonists, CNQX and D-AP5, partially reduced the ANG-(1-7) effect. ATP is often co-released with glutamate, and blocking ATP with PPADS also reduced the pressor response to microinjection of ANG-(1-7) within the RVLM. The effects of ANG-(1-7) were blocked by the MAS receptor antagonist, A-779 (which had no consistent effect on blood pressure or sympathetic nerve activity when injected on its own). We conclude that astrocytes in the RVLM participate in central, angiotensin-dependent regulation of blood pressure and sympathetic nerve activity, and the Mas receptor, when activated by ANG-(1-7), elicits the release of the gliotransmitters, glutamate and ATP. These gliotransmitters then cause an increase in sympathetic nerve activity and blood pressure by interacting with AMPA/kainate and P2X receptors in the RVLM.

The efficiency of bovine in vitro embryo production has remained low despite extensive effort to understand the effects of culture conditions, media composition and supplementation. As bovine oocytes resume meiosis spontaneously when cultured, it was hypothesized that preventing meiosis in vitro before in vitro maturation (IVM) and in vitro fertilization (IVF) would allow more oocytes to acquire developmental competence. This article reviews some of the factors involved in meiotic arrest as well as the effects of meiotic inhibition before IVM on bovine oocytes developmental competence following IVF. Follicular components and cAMP-elevating agents can delay or inhibit meiosis in various proportions of oocytes; however, few studies have examined their effects on development following IVM and IVF because they are not practical (follicular components) or have a transient effect on meiosis (cAMP-elevating agents). Protein synthesis or phosphorylation inhibition prevented meiosis in high percentages of oocytes; however, these non-specific inhibitions led to lower developmental competence compared with non-arrested oocytes. Maturation promoting factor (MPF) inhibition with specific inhibitors has been examined in several studies. Despite faster maturation following removal from inhibition and some structural damage to the oocytes, MPF inhibition generally led to blastocyst rates similar to control, non-arrested oocytes. Future work will involve evaluating the effects on arrested oocytes of molecules that can improve developmental competence in non-arrested oocytes. It is also anticipated that new IVM systems that take into consideration new knowledge of the mechanisms involved in the control of meiosis will be developed. Moreover, global gene expression analysis studies will also provide clues to the culture conditions required for optimal expression of developmental competence.

There is a need for better understanding of the biocompatibility of alginate-polycation microcapsules based on their physicochemical characteristics. Microcapsules composed of alginate with 44% (IntG) or 71% (HiG) guluronate, gelled with calcium (Ca) or barium (Ba) and coated with poly-L-lysine (PLL) or poly-l-ornithine (PLO), followed by IntG alginate were compared. For microcapsules with an IntG(Ca) gel core, using PLO instead of PLL resulted in less immune cell adhesion after 2 days in C57BL/6J mice. The PLO microcapsules were also characterized by greater hydrophilicity and superior resistance to swelling and damage under osmotic stress. For microcapsules with a PLL membrane, replacing the IntG(Ca) gel core with IntG(Ba) or HiG(Ca) gel resulted in stronger immune responses (p

Alginate purification has been shown to decrease the host immune response to implanted alginate-based microcapsules, but the direct effect of contaminants on islet cell survival remains unknown. Wistar rat islets were immobilized in calcium alginate beads made with crude vs. purified alginate and then incubated in CMRL culture medium. Islet survival was evaluated at 1, 4, 7, 14 and 27 days post-encapsulation. Islet viability was investigated using a dual staining assay (propidium iodide and orange acridine). The islet cell necrosis and the proportion of apoptotic cells were quantified under optical microscopy and with a TUNEL assay, respectively. Islets immobilized in purified alginate were more viable, and had fewer necrotic centers, a smaller area of central necrosis and a lower number of apoptotic cells. At day 14 and 27 post-encapsulation, respectively, 48% and 23% of islets were viable with purified alginate vs. 18% and 8% with crude alginate (p

Meiotic maturation in mammalian oocytes is initiated during fetal development, arrested for several years in some cases, then completed at the time of ovulation. Although the mechanisms involved in oocyte meiotic arrest and meiotic resumption are not fully understood, new players and roles have recently been identified. This paper reviews the role of follicle cells, as well as the role of the cyclic adenosine 3', 5'-monophosphate (cAMP) pathway, in the maintenance of meiotic arrest. Potential mechanisms by which luteinizing hormone (LH) signals meiotic resumption are also reviewed. New findings on the role of adenosine monophosphate-activated kinase (PRKA), as well as the effects of culture medium composition on meiosis in vitro, are also discussed. From a practical perspective, improved understanding of the mechanisms involved in the control of meiosis will facilitate better control of the process in vitro resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures. Key words: Bovine, oocyte, meiosis, cAMP

Ubiquitination functions as a sorting signal for lysosomal degradation of cell-surface proteins by facilitating their internalization from the plasma membrane and incorporation into lumenal vesicles of multivesicular bodies (MVBs)1. Ubiquitin may also mediate sorting of proteins from the trans-Golgi network (TGN) to the endosome, thereby preventing their appearance on the cell surface and hastening their degradation in the lysosome–vacuole2,3,4,5,6. Substantiation of a direct ubiquitin-dependent TGN sorting pathway relies in part on identifying candidate machinery that may function as a ubiquitin-sorting 'receptor'at the TGN. Members of the GGA family of coat proteins localize to the TGN and promote the incorporation of proteins into clathrin-coated vesicles destined for transport to endosomes7,8. We show that the GGA coat proteins bind directly to ubiquitin through their GAT domain and demonstrate that this interaction is required for the ubiquitin-dependent sorting of the Gap1 amino acid transporter from the TGN to endosomes. Thus, GGA proteins fulfill the role of ubiquitin sorting receptors at the TGN.

Embryonic mortality in cattle is the death of the conceptus before Day 42 of pregnancy. Estimates of embryonic mortality varied greatly, ranging from 10 to 40% in first-service cattle to 65% in repeat-breeder cows. This paper reviews endogenous causes of embryonic mortality, including chromosomal abnormalities, inadequate corpus luteum function and the influence of follicular development on fertility. External causes of embryonic mortality, including heat stress and nutrition, are also reviewed; these factors act by modifying the follicular environment (thereby affecting oocyte quality) or by creating a hostile uterine environment. Strategies to reduce embryonic mortality, including good management practices and supplementation with progesterone or interferon-tau, are discussed. From a practical perspective, improved knowledge of the causes of embryonic mortality and regulation of follicular growth and corpus luteum function should result in the development of improved, cost-effective hormonal regimes that effectively synchronize estrus and ovulation and reduce embryonic mortality, thereby benefiting the cattle industry. Key words: Cattle, reproduction, fertility, embryo development, embryo mortality

Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting “receptors,” the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.

Membrane proteins that are degraded in the vacuole of Saccharomyces cerevisiae are sorted into discrete intralumenal vesicles, analogous to the internal membranes of multi-vesiculated bodies (MVBs). Recently, it has shown that the attachment of ubiquitin (Ub) mediates sorting into lumenal membranes. We describe a complex of Vps27p and Hse1p that localizes to endosomal compartments and is required for the recycling of Golgi proteins, formation of lumenal membranes and sorting of ubiquitinated proteins into those membranes. The Vps27p-Hse1p complex binds to Ub and requires multiple Ub Interaction Motifs (UIMs). Mutation of these motifs results in specific defects in the sorting of ubiquitinated proteins into the vacuolar lumen. However, the recycling of Golgi proteins and the generation of lumenal membranes proceeds normally in Delta UIM mutants. These data support a model in which the Vps27p-Hse1p complex has multiple functions at the endosome, one of which is as a sorting receptor for ubiquitinated membrane proteins destined for degradation.

The developmental potential of adult somatic nuclei after nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated in a dwarf breed of goat (BELE: Breed Early Lactate Early). Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) fetal fibroblasts. Primary GCs were obtained from follicular aspirants after laparoscopic oocyte pick-up (LOPU) and were cryopreserved immediately. Frozen aliquots of cells were thawed and cultured until confluent and were then cultured in low serum for 4 days before use in NT. Immature oocytes were obtained by LOPU and matured before enucleation and NT. Ninety-one adult GC-derived NT embryos were transferred into eight recipients, four of which were confirmed pregnant (50%) at Day 30 by ultrasound. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients, one of which was confirmed pregnant (17%). All pregnancies were maintained through term. Four recipients delivered seven female kids (three sets of twins) derived from the GC cultures (7.7% of embryos transferred). The other recipient delivered two male kids (3.7% of embryos transferred). Birth weights were within the normal range for dwarf goats. One female twin and one male twin died at birth; the remaining kids appeared healthy and normal. DNA analysis confirmed that the kids were genetically identical to their respective donors. These results demonstrated that adult caprine somatic cells could direct normal development after NT.

The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3′ splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1 B , A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

We isolated a revertant virus after prolonged culturing of a replication-impaired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG translation initiation codon. Sequencing of the tat - rev region of this revertant virus identified a second-site mutation in tat that restored virus replication in the mutant background. This mutation activated a cryptic 5′ splice site (ss) that, when used in conjunction with the regular HIV 3′ ss #5, fuses the tat and rev reading frames to encode a novel T-Rev fusion protein that rescues Rev function. We also demonstrate an alternative route to indirectly activate this cryptic 5′ ss by mutational inactivation of an adjacent exon splicing silencer element.

The goal of prosthetic rehabilitation is to compensate for the loss of a limb by amputation by, in the case of a lower limb, encouraging walking, and to achieve the same level of autonomy as prior to the amputation. However, because of difficulties walking, elderly amputees may use their prosthesis to a greater or lesser degree or simply stop using it during the rehabilitation period. The objective of this research was to study factors such as physical and mental health, rehabilitation, physical independence and satisfaction with the prosthesis to understand why amputees use their prosthesis or not. The sample was composed of 65 unilateral vascular amputees 60 years old or over living at home. The information was collected from medical records, by telephone interview and by mail questionnaire. Prosthesis use was measured by a questionnaire on amputee activities developed by Day (1981). Eighty-one per cent (81%) of the subjects wore their prosthesis every day and 89% of this group wore it 6 hours or more per day. Less use of the prosthesis was significantly related to age, female gender, possession of a wheelchair, level of physical disability, cognitive impairment, poorer self-perceived health and the amputee's dissatisfaction. A multiple regression analysis showed that satisfaction, not possessing a wheelchair and cognitive integrity explained 46% of the variance in prosthesis use.

Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus that transcribes its RNA from an integrated proviral genome and uses the host splicing machinery to produce its mRNAs. To generate the more than 30 different singly and multiply spliced mRNAs, the HIV-1 9.2-kb primary transcript undergoes splicing by a complex pathway. The regulatory proteins Tat, Rev, and Nef are encoded by multiply spliced ∼2-kb mRNAs, whereas the Env, Vif, Vpr, and Vpu proteins are encoded by singly spliced mRNAs. In addition, approximately half of the HIV-1 RNA remains unspliced and is used as message for gag and pol gene products. The unspliced RNA is also packaged into progeny virions. The HIV-1 Rev protein, which binds to the Rev-responsive element located in the env gene, facilitates the stabilization and nuclear transport of unspliced and partially spliced mRNAs. However, prior to the action of Rev, the efficiency of viral RNA splicing at the different splice sites is regulated by a number of cis elements within the viral genome. Several splicing elements have been localized within the first tat coding exon of the NL4-3 HIV-1 strain (tat exon 2). One of these elements, has been termed exon splicing silencer 2 (ESS2), is located 60 to 70 nucleotides (nt) downstream from the 3′ splice site (3′ splice site A3) used to generate Tat mRNA (Fig. ​(Fig.1A)1A) (3, 4). It maps to the 10-nt core sequence CUAGACUAGA and acts specifically to inhibit splicing at this 3′ splice site (18). Mutations within ESS2 result in a selective increase in tat splicing when tested in an in vitro splicing system or in cell culture after transfection of infectious proviral DNA or virus infection (3, 7, 18). Inspection of HIV-1 sequences in the database (9a) indicates that the region of ESS2 in many strains within the M (major) group of HIV-1 strains contain only one copy of the CUAGA sequence, which is repeated in the NL4-3 ESS2. The corresponding sequences of the O (outlier) group of HIV-1 strains are more divergent and do not contain even a single copy of the CUAGA sequence, which suggests that there may be significant differences in the regulation of tat splicing in different HIV strains. FIG. 1 RT-PCR analysis of multiply spliced HIV-mRNAs in cells transfected with wild-type and minigene NL4-3 constructs. (A) Structure of the NL4-3 HIV-1 genome. Locations of the known 5′ (D) and 3′ (A) splice sites (ss) and the ESS2 are shown. ... A second splicing element in tat exon 2 is the most 3′-terminal of the three AG dinucleotides used to generate rev mRNAs (3′ splice site A4b [Fig. 1A]). We have shown that the A4b AG serves as both a splice site for rev mRNA and a branchpoint sequence (BPS) for splicing of single-spliced and double-spliced mRNAs encoding the Env and Nef proteins, respectively (22). Mutagenesis of the A4b AG results in significantly increased splicing at the env/nef 3′ splice site (A5) and a concomitant replication defect (16, 22). We have hypothesized that the increase in splicing at A5 results from relief of competition for AG binding factors. The consequence of this is facilitated binding of a factor(s) to the branchpoint. The A4b 3′ splice site is conserved in all group M HIV-1 strains but not in any group O strains. However, in a fraction of group M and all group O strains, some of the other rev AG dinucleotides are not present and different AG dinucleotides are created that are potential new rev splice sites. These sequence differences among the HIV-1 strains suggest that regulation of splicing may be different in these strains. To test for this, we have compared alternative splice site usage of NL4-3 in the region of the first tat coding exon to two other virus strains: (i) a diverged group M, clade B virus (SF2 strain) with changes in the ESS2 sequence as well as in the region of the rev 3′ splice sites and (ii) a highly divergent group O virus strain (ANT70C). Our results indicate that regulatory elements in the region of tat exon 2 are characteristic of group M but not group O viruses.

Frost formation in rotary heat and mass exchangers is investigated both experimentally and numerically. Experiments reveal that glazed frost, whose density and thermal conductivity are larger than that of rough frost, prevails in rotary exchangers operating in cold climates. A mathematical model for the prediction of the thermal behavior of the exchanger is presented. The model is validated with experimental data and employed to conduct a parametric study. Results indicate that the absolute humidity is the prevailing parameter to characterize the frosting phenomenon. A frost mass fraction chart is established in terms of the relative humidity of the warm exhaust stream and of the temperature of the cold supply stream. The transient three-dimensional model also shows that the absolute humidity and the temperature of both air flows vary nonlinearly in the frosted zone.

With the diabetes epidemic affecting the world population, there is an increasing demand for means to regulate glycemia. Dietary glucose is first absorbed by the intestine before entering the blood stream. Thus, the regulation of glucose absorption by intestinal epithelial cells (IECs) could represent a way to regulate glycemia. Among the molecules involved in glycemia homeostasis, extracellular ATP, a paracrine signaling molecule, was reported to induce insulin secretion from pancreatic β cells by activating P2Y and P2X receptors. In rat's jejunum, P2X7 expression was previously immunolocalized to the apex of villi, where it has been suspected to play a role in apoptosis. However, using an antibody recognizing the receptor extracellular domain and thus most of the P2X7 isoforms, we showed that expression of this receptor is apparent in the top two-thirds of villi. These data suggest a different role for this receptor in IECs. Using the non-cancerous IEC-6 cells and differentiated Caco-2 cells, glucose transport was reduced by more than 30% following P2X7 stimulation. This effect on glucose transport was not due to P2X7-induced cell apoptosis, but rather was the consequence of glucose transporter 2 (Glut2)'s internalization. The signaling pathway leading to P2X7-dependent Glut2 internalization involved the calcium-independent activation of phospholipase Cγ1 (PLCγ1), PKCδ, and PKD1. Although the complete mechanism regulating Glut2 internalization following P2X7 activation is not fully understood, modulation of P2X7 receptor activation could represent an interesting approach to regulate intestinal glucose absorption. J. Cell. Physiol. 228: 120–129, 2013. © 2012 Wiley Periodicals, Inc.

Several lines of evidence indicate that calcium deficiency is associated with cellular defects in many tissues and organs. Owing to the large in vivo gradient between ionized extra- and intracellular Ca2+ concentrations ([Ca2+]i), it is generally recognized that the prevailing circulating Ca2+ does not significantly affect resting cytosolic Ca2+. To probe the consequences of hypocalcemia on [Ca2+]i, a model of chronic hypocalcemia secondary to vitamin D (D) deficiency was used. Hepatocytes were isolated from livers of hypocalcemic D-deficient, of normocalcemic D3-repleted, or of normal control rats presenting serum Ca2+ of 0.78 +/- 0.02, 1.24 +/- 0.03, or 1.25 +/- 0.01 mM, respectively (P < 0.0001). [Ca2+]i was measured in cell couplets using the fluorescent probe Fura-2. Hepatocytes of normocalcemic D3-repleted and of normal controls exhibited similar [Ca2+]i of 227 +/- 10 and 242 +/- 9 nM, respectively (NS), whereas those of hypocalcemic rats had significantly lower resting [Ca2+]i (172 +/- 10 nM; P < 0.0003). Stimulation of hepatocytes with the alpha 1-adrenoreceptor agonist phenylephrine illicited increases in cytosolic Ca2+ leading to similar [Ca2+]i and phosphorylase a (a Ca(2+)-dependent enzyme) activity in all groups but in contrast to normocalcemia, low extracellular Ca2+ was often accompanied by a rapid decay in the sustained phase of the [Ca2+]i response. When stimulated with the powerful hepatic mitogen epidermal growth factor (EGF), hepatocytes isolated from hypocalcemic rat livers responded with a blunted maximal [Ca2+]i of 237.6 +/- 18.7 compared with 605.2 +/- 89.9 nM (P < 0.0001) for their normal counterparts, while the EGF-mediated DNA synthesis response was reduced by 50% by the hypocalcemic condition (P < 0.03). Further studies on the possible mechanisms involved in the perturbed [Ca2+]i homeostasis associated with chronic hypocalcemia revealed the presence of an unchanged plasma membrane Ca2+ ATPase but of a significant decrease in agonist-stimulated Ca2+ entry as indicated using Mn2+ as surrogate ion (P < 0.03). Our data, thus indicate that, in rat hepatocytes, the in vivo calcium status significantly affects resting [Ca2+]i, and from this we raise the hypothesis that this lower than normal [Ca2+]i may be linked, in calcium disorders, to inappropriate cell responses mediated through the calcium signaling pathway as illustrated by the response to phenylephrine and EGF.

Background: Inflammatory bowel diseases are characterized by the presence of CXCL8 at the site of lesions resulting in neutrophil recruitment and loss of tissue functions. We report that P2Y6 receptor activation stimulates CXCL8 expression and release by intestinal epithelial cells (IECs). In this context, we investigated if uridine 5′-diphosphate (UDP) enemas stimulate neutrophil recruitment to the mucosa of mice suffering from colitis-like disease and we characterized the signaling events linking P2Y6 to CXCL8 expression in IEC. Methods: Neutrophil recruitment was monitored by immunofluorescence and FACS analysis. Expression of Cxcl1, a mouse functional homolog of CXCL8, was determined by quantitative real-time polymerase chain reaction (qPCR). Pharmacological inhibitors and interfering RNAs were used to characterize the signaling pathway. The outcomes of these treatments on protein phosphorylation and on CXCL8 expression were characterized by western blots, qPCR, luciferase, and chromatin immunoprecipitation (ChIP) assays. Results: Mutation of the AP-1 site in the CXCL8 core promoter abolished the UDP-stimulating effect. The c-fos/c-jun dimer was identified as the AP-1 complex regulating CXCL8 in response to UDP stimulation. Regulation of CXCL8 expression by P2Y6 required PKCδ activation upstream of the signaling pathway composed of MEK1/2-ERK1/2 and c-fos. UDP administration to mice suffering from colitis-like disease increased the number of neutrophil infiltrating the mucosa, correlating with Cxcl1 increased expression in IEC and the severity of inflammation. Conclusions: This study not only describes the P2Y6 signaling mechanism regulating CXCL8 expression in IEC, but it also illustrates the potential of targeting P2Y6 to reduce intestinal inflammation. (Inflamm Bowel Dis 2012)

Alginate remains the most popular polymer used for cell encapsulation, yet its biocompatibility is inconsistent. Two commercially available alginates were compared, one with 71% guluronate (HiG), and the other with 44% (IntG). Both alginates were purified, and their purities were verified. After 2 days in the peritoneal cavity of C57BL/6J mice, barium (Ba)-gel and calcium (Ca)-gel beads of IntG alginate were clean, while host cells were adhered to beads of HiG alginate. IntG gel beads, however, showed fragmentation in vivo while HiG gel beads stayed firm. The physicochemical properties of the sodium alginates and their gels were thoroughly characterized. The intrinsic viscosity of IntG alginate was 2.5-fold higher than that of HiG alginate, suggesting a greater molecular mass. X-ray photoelectron spectroscopy indicated that both alginates were similar in elemental composition, including low levels of counterions in all gels. The wettabilities of the alginates and gels were also identical, as measured by contact angles of water on dry films. Ba-gel beads of HiG alginate resisted swelling and degradation when immersed in water, much more than the other gel beads. These results suggest that the main factors contributing to the biocompatibility of gels of purified alginate are the mannuronate/guluronate content and/or intrinsic viscosity.

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat β-casein promoter. Milk from transgenic animals contained 0.1–5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.

Background Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice. Results The GUM-GFP cassette was constructed and transgenic mice were generated in order to study the promoter's tissue specificity, the GFP kidney specific expression and its subcellular distribution. Tissues collected from three GUM-GFP transgenic mouse lines, and analyzed for the presence of GFP by Western blotting and fluorescence confirmed that the GUM promoter drove expression of GFP specifically in the kidney. More specifically, by using immuno-histochemistry analysis of kidney sections, we demonstrated that GFP expression was co-localized, with endogenous uromodulin protein, in the epithelial cells of the thick ascending limbs (TAL) of Henle's loop and the early distal convoluted tubule in the kidney. Conclusion The goat uromodulin promoter is capable of driving recombinant protein expression in the kidney of transgenic mice. The goat promoter fragment cloned may be a useful tool in targeting proteins or oncogenes in the kidney of mammals.

In vitro transfection of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. In the present study, bovine primary fetal fibroblasts were transfected with a green fluorescent protein (GFP)-reporter transgene and used as nuclear donor cells in oocyte reconstructions. Because cell synchronization protocols are less effective after transfection, activated oocytes may be more suitable as hosts for nuclear transfer. To examine the role of host cytoplasm on transgene expression and developmental outcome, GFP-expressing fibroblasts were fused to oocytes reconstructed either before (metaphase) or after (telophase) activation. Expression of GFP was examined during early embryogenesis, in tissues of cloned calves, and again during embryogenesis, after passage through germ line using semen from the transgenic cloned offspring. Regardless of the kind of host cytoplasm used, GFP became detectable at the 8- to 16-cell stage, approximately 80 h after reconstruction, and remained positive at all later stages. After birth, although cloned calves obtained through both procedures expressed GFP in all tissues examined, expression levels varied both between tissues and between cells within the same tissue, indicating a partial shutdown of GFP expression during cellular differentiation. Moreover, nonexpressing fibroblasts derived from transgenic offspring were unable to direct GFP expression after nuclear transfer and development to the blastocyst stage, suggesting an irreversible silencing of transgenes. Nonetheless, GFP was expressed in approximately half the blastocysts obtained with sperm from a transgenic clone, confirming transmission of the transgene through the germ line.

The complex oxide dielectric Ba/sub 1-x/Sr/sub x/TiO/sub 3/ (BST) has recently been studied intensively for DRAM applications. Another significant nearer term application for which BST may be suited is replacement of discrete capacitors now attached to advanced IC's. Specific devices include bypass, decoupling, and switched filter capacitors, often operated at high frequencies. With current SiO/sub 2/ based dielectrics such integrated capacitors may take up from 20% to 50% of the device area; use of higher dielectric constant BST could reduce this by at least a factor of 10. We will discuss properties of MOCVD-grown BST which relate to these capacitor applications, including second order dielectric nonlinearity and leakage currents at useful device operating voltages (3 V). Dielectric constant, nonlinearity and breakdown voltages are found to have a strong dependence on deposition temperature of BST films, so optimum processing conditions will depend on the end device application.

The developmental potential of adult somatic nuclei after nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated in a dwarf breed of goat (BELE: Breed Early Lactate Early). Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) fetal fibroblasts. Primary GCs were obtained from follicular aspirants after laparoscopic oocyte pick-up (LOPU) and were cryopreserved immediately. Frozen aliquots of cells were thawed and cultured until confluent and were then cultured in low serum for 4 days before use in NT. Immature oocytes were obtained by LOPU and matured before enucleation and NT. Ninety-one adult GC-derived NT embryos were transferred into eight recipients, four of which were confirmed pregnant (50%) at Day 30 by ultrasound. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients, one of which was confirmed pregnant (17%). All pregnancies were maintained through term. Four recipients delivered seven female kids (three sets of twins) derived from the GC cultures (7.7% of embryos transferred). The other recipient delivered two male kids (3.7% of embryos transferred). Birth weights were within the normal range for dwarf goats. One female twin and one male twin died at birth; the remaining kids appeared healthy and normal. DNA analysis confirmed that the kids were genetically identical to their respective donors. These results demonstrated that adult caprine somatic cells could direct normal development after NT.

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

233 (score 1.02 ± 0.03). This difference in biocompatibility was significant (p < 0.001). Yet, alginate hydrophilicity was similar, as water droplets on dry films had contact angles of 38.7 ± 1.7° and 39.0 ± 1.3° for Alg I and Alg II, respectively. When PLL was added to the capsule membrane, the immune response was more severe (p < 0.01), as cell adhesion scores increased to 1.11 ± 0.11 for Alg I and to 1.55 ± 0.07 for Alg II. Similarly, the cell adhesion score increased to 0.49 ± 0.06 when PLO was added to Alg I capsules, indicating a non-significant increase in capsule immunogenicity. A comparison of contact angles of water on dry films containing polycations was not possible due to irregular surfaces and complete wetting. We are currently measuring the contact angles of air bubbles on hydrated films. conclusions: The guluronic acid content of alginate has a significant effect on its biocompatibility in vivo. Incorporating polycations into the membranes of alginate microcapsules lowers their biocompatibility. So far, these results cannot be explained by the hydrophilicity of the samples. Transplantation islet and pancreas No conflict of interest

The satisfaction of persons with lower-limb amputations towards their prosthesis constitutes a critical factor in the use of the prosthesis. In order to evaluate a person's satisfaction, the SAT-PRO, a self-administrated questionnaire was developed. The questionnaire includes 15 items developed on the basis of the most significant criteria used by the person when selecting a technical aid. These criteria were measured using an ordinal categorical four-level scale. The validation of the SAT-PRO was established from a sample consisting of 61 people with below-knee or above-knee amputations, aging from 60 years and older. The internal consistency of the instrument is high (Cronback's alpha coefficient is 0.90) and the test-retest reliability coefficient (0.97) indicates a very good consistency of the questionnaire over time. Simple and multiple correlations were used to evaluate construct validity. The degree of use of the prosthesis and the feelings of depression, amongst the measured variables, are the best indicators of the satisfaction of the amputees toward their prosthesis.

The relative abundances of transcripts of different origins and housekeeping functions were measured by Northern blot analysis of RNA samples derived from in vitro-matured oocytes and in vitro-produced bovine embryos at selected stages of early development. The gene products studied included: two mitochondrial transcripts, 12S rRNA and cytochrome b mRNA; two RNAs involved in the processing of other RNAs, U2 and U3 snRNA; and two nuclear-derived transcripts, beta-actin mRNA and histone H3 mRNA. Overall, the RNA levels for the various genes studied remained constant or decreased slightly from the mature oocyte to the 6- to 8-cell or morula stage and were greatly increased in blastocysts. Differences were observed in the degree to which the RNA levels increased and in the timing of the increase. For 12S rRNA, a major increase was not observed until the blastocyst stage where levels increased 7.1 times the amount detected in morulae. Cytochrome b mRNA levels started to increase at the 6- to 8-cell stage and reached levels in blastocysts that were 20 times more than the cytochrome b mRNA level in 2- to 4-cell embryos. U2 snRNA levels did not increase until the blastocyst stage where levels were 6.4 times the amount found in morulae. U3 snRNA and beta-actin mRNA levels started to increase at the morula stage and blastocysts contained 118 and 110 times more U3 snRNA and beta-actin mRNA, respectively, than 6- to 8-cell embryos. However, blastocysts contained only two times the amount of histone H3 mRNA present in 6- to 8-cell embryos.

The abundance of 28S, 18S, and 5S rRNA was measured by Northern blot techniques applied to RNA samples extracted from bovine oocytes and preattachment embryos produced by in vitro procedures. Total RNA content was estimated by comparing the intensity of hybridization signals of 28S and 18S rRNA probes to embryo RNA samples and to standard curves generated from bovine ovary or bovine oviduct cell RNA. RNA content declined from the oocyte to the morula stage (2.4 +/- 0.3 ng/oocyte, 1.7 +/- 0.5 ng/1-cell embryo, 2.2 +/- 0.9 ng/2- to 4-cell embryo, 0.8 +/- 0.2 ng/6- to 8-cell embryo, and 0.7 +/- 0.2 ng/morula). A marked increase in RNA content, based on levels of hybridization to 28S and 18S rRNA, was observed in blastocysts, in which values averaged 5.3 +/- 0.6 ng/embryo. On a relative basis, 5S rRNA abundance followed a pattern similar to that of 28S and 18S rRNA across the early development period to the blastocyst stage.

To determine the association between mitral regurgitation and pulmonary edema localized in the right upper lobe, the authors reviewed 21 cases of mitral regurgitation secondary to dysfunction or rupture of the papillary muscle or rupture of the chordae tendineae cordis. The patients, 12 men and 9 women ranging in age from 36 to 92 (mean 64) years, had been admitted to a tertiary care hospital between July 1985 and July 1990. Three independent observers, who were unaware of the patients' identity or the diagnoses, reviewed the chest radiographs. In eight of the patients pulmonary edema was localized preferentially in the right upper lobe, an unusual pattern that can simulate neoplasia, hemorrhage or infection. All eight patients had myocardial infarction, five had papillary muscle dysfunction, and three had rupture of the posterior papillary muscle. Mitral regurgitation toward the orifices of the veins of the right upper lobe seems to play a role in the preferential distribution of edema to that lobe. Awareness of edema in the right upper lobe in association with mitral regurgitation might lead to earlier diagnosis of papillary rupture or dysfunction and perhaps affect the outcome.

The throughput of a grazing incidence optical system is highly dependent on the reflectivities of the coatings used. To support ongoing work at HDOS for the Advanced X-ray Astrophysics Facility, AXAF, we have begun to look at materials and processes which maximize reflectivity in the 1-10 keV range. These processes must be scaleable and compatible with the coating of the AXAF elements. These elements are concentric paraboloids and hyperboloids ranging in diameter from 18 to 40 inches with an axial length of 40 inches. The coatings are applied to the interior optical surfaces.

Islet transplantation has normalized the blood glucose levels in patients with type 1 diabetes. However, this treatment requires the use of potentially harmful immunosuppressive drugs. An alternative to immunosuppression is the use of semi-permeable microcapsules that allow the diffusion of glucose, nutrients and oxygen but protect the transplanted cells against immune reaction. Another important problem is the limited survival of encapsulated and free islet cells. Pancreatic duct cells have been shown to play an important role in pancreas morphogenesis. The co-incubation of pancreatic duct cells and islets has decreased cell apoptosis and necrosis in the immediate post-isolation period. We have previously shown that incubating islets with IGF-II, the principal peptide that is secreted by duct cells, diminishes apoptosis and necrosis of encapsulated islets and allows the restoration of euglycaemia in diabetic mice using fewer islets. The objective of this study is to investigate the effect of co-encapsulating islets with pancreatic duct cells on islet cell survival. Pancreatic duct cells were isolated from C57BL/6 mice and their identity was confirmed by immunohistochemistry using antibodies against cytokeratin 7 (CK7) and IGF-II. The presence of known contaminants (fibroblasts and mesenchymals cells) was investigated by flow cytometry using their common marker: vimentin. Islets encapsulated alone or co-encapsulated with two different concentrations of duct cells (20 or 100 cells per capsule) were maintained in culture and evaluated at days 1, 7, 14 and 27 postencapsulation. Islet viability was evaluated using fluorescent dyes (acridine orange and propidium iodide), the percentage of necrotic islets by inverted-microscope analysis and their function by an MTS test. Duct cells expressed IGF-II and CK7. The cell preparation was pure at >85%. Islets cell viability was between 60-80% for islets encapsulated alone or with duct cells with no significant difference. This coencapsulation did not have a significant impact on the islets necrosis which remained consistently between 30-40%. Co-encapsulating islets with 100 duct cells had a significant impact on islet function (vs. islets encapsulated alone). This was demonstrated by the MTS test, which showed an absorbance at 492 nm of 0.32 ± 0.06 for co-encapsulated islets (vs 0.09 ± 0.01; p

The oocytes of cattle are not as sensitive as those of laboratory animals to purines, cAMP, or follicular extracts. To study the resumption of meiosis, a method is needed that is capable of inhibiting meiosis completely for a minimum of 24 h. This study was designed to evaluate interrelationships in granulosa-oocyte-cumulus complexes using fresh granulosa cells aspirated from small follicles (1-5 mm) in which the cumulus is normally firmly attached. Selected oocyte-cumulus complexes obtained from a slaughterhouse (n = 2,236) were co-incubated with one of the following: various concentrations of fresh granulosa cells in tissue cultures medium (TCM) 199 or bovine follicular fluid (BFF) either without or after one washing and/or freezing; resuspended granulosa cells previously cultured for 7 days; blood cells; or medium alone. Additionally, oocyte-cumulus complexes were embedded in agar cylinders before incubation with or without cells. The rate of maintenance of intact germinal vesicles (GV) in oocytes after 24 h ranged from 40-77% when 5-100 x 10(6) unwashed cells/ml BFF were used, compared to only 16% in oocytes cultured in BFF alone. The pattern was the same when washed cells were used (30-77%, using 5-100 x 10(6) cells/ml BFF), but they were not as effective as unwashed cells. With TCM-199 and the same five concentrations of cells (5, 10, 25, 50, and 100 x 10(6)/ml), a similar inhibition was obtained with greater than or equal to 25 but not with 5 (3%) or 10 (5%) x 10(6)/ml.(ABSTRACT TRUNCATED AT 250 WORDS)