Structural arrangement of the VH and VL domains in the COBRA™ T-cell engaging single chain diabody

Background COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding VH and VL and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi, into scFvs is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired. Methods We analyzed the structure of an EGFR COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS). Results We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Non-enzymatic post-translational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites. Conclusion Here we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.

Cyclic adenosine monophosphate modulator supplementation on in vitro maturation of bovine oocytes

Study evaluated the role of cAMP modulator (Forskolin and 3-isobutyl-1- methyl xanthine) supplementation on developmental competence of bovine oocytes. Cumulus oocyte complexes recovered from bovine ovaries of unknown reproductive status were used for the study. Oocytes retrieved by aspiration method were graded based on cumulus cell distribution and culture grade oocytes were selected for the study. A total of 414 culture grade oocytes were taken and divided into two groups. Group I constituted of 201 oocytes in which pre-maturation was carried out for a period of 2 h. In group II, 213 oocytes were selected in which normal maturation was carried out. Maturation was assessed after 24h of culture in CO2 incubator maintained at 38.5o C in 95 per cent humidified atmosphere of 5 per cent CO2 . Fertilisation was carried out using frozen thawed semen and the presumptive zygotes were then transferred to culture media and cleavage was assessed 48 h after insemination. A significantly higher maturation rate (p ?0.05) was observed in group I compared to group II (86.85 ± 1.19 vs 79.88 ± 2.67). There was a highly significant increase (p ?0.01) in cleavage rate in group I (65.92 ± 1.23) compared to group II (59.29 ± 1.50). A higher fertilisation rate was observed in group I (75.35 ± 1.19) than group II (71.88 ± 2.56). It could be concluded that pre-maturation with cAMP modulators improved the developmental competence of bovine oocytes.

Grape Seed Proanthocyanidin Ameliorates FB1-Induced Meiotic Defects in Porcine Oocytes

Fumonisin B1 (FB1), as the most prevalent and toxic fumonisin, poses a health threat to humans and animals. The cytotoxicity of FB1 is closely related to oxidative stress and apoptosis. The purpose of this study is to explore whether Grape seed proanthocyanidin (GSP), a natural antioxidant, could alleviate the meiotic maturation defects of oocytes caused by FB1 exposure. Porcine cumulus oocyte complexes (COCs) were treated with 30 μM FB1 alone or cotreated with 100, 200 and 300 μM GSP during in vitro maturation for 44 h. The results show that 200 μM GSP cotreatment observably ameliorated the toxic effects of FB1 exposure, showing to be promoting first polar body extrusion and improving the subsequent cleavage rate and blastocyst development rate. Moreover, 200 μM GSP cotreatment restored cell cycle progression, reduced the proportion of aberrant spindles, improved actin distribution and protected mitochondrial function in FB1-exposed oocytes. Furthermore, reactive oxygen species (ROS) generation was significantly decreased and the mRNA levels of CAT, SOD2 and GSH-PX were obviously increased in the 200 μM GSP cotreatment group. Notably, the incidence of early apoptosis and autophagy level were also significantly decreased after GSP cotreatment and the mRNA expression levels of BAX, CASPASE3, LC3 and ATG5 were markedly decreased, whereas BCL2 and mTOR were observably increased in the oocytes after GSP cotreatment. Together, these results indicate that GSP could exert significant preventive effects on FB1-induced oocyte defects by ameliorating oxidative stress through repairing mitochondrial dysfunction.

Effect of enzymatic pro-oxidant and antioxidant systems on bovine oocyte in vitro maturation

The role of reactive oxygen species (ROS) during oocyte in vitro maturation (IVM) is still controversial. Although an increase in ROS production may cause deleterious effects in cells, these reactive species may also act as signaling molecules influencing different cell functions. The aim of this study was to examine the effect of varying endogenous ROS levels during IVM on the process of bovine oocyte maturation. To do so, different enzymatic antioxidant (catalase, or superoxide dismutase + catalase, or diphenyl iodonium) or pro-oxidant systems (xanthine + xanthine oxidase, or xanthine + xanthine oxidase + catalase) were added to the culture medium. ROS levels were determined by 2′,7′-dichlorodihydrofluorescein diacetate stain, nuclear maturation was evaluated by the presence of the metaphase II chromosome configuration at 22h of IVM and cleavage rate was recorded 48hs post- in vitro fertilization. ROS levels were only significantly increased (P<0.05) by the O2 .- generating system (xanthine + xanthine oxidase + catalase), but meiotic maturation rates were significantly lower (P<0.05) in all the evaluated systems compared with the control, except for the diphenyl iodonium group. However, this last group presented a significantly lower (P<0.05) cleavage rate in comparison to the control group. These results indicate that ROS would play an essential role during oocyte maturation, since its increase or decrease beyond a physiological level significantly reduced nuclear or cytoplasmic maturation rates in bovine oocytes.

GSK3640254 Is a Novel HIV-1 Maturation Inhibitor With an Optimized Virology Profile

HIV-1 maturation inhibitors (MIs) offer a novel mechanism of action and potential for use in HIV-1 treatment. Prior MIs displayed clinical efficacy but were associated with the emergence of resistance and some gastrointestinal tolerability events. Treatment with the potentially safer next-generation MI GSK3640254 (GSK’254) resulted in up to a 2-log 10 viral load reduction in a phase IIa proof-of-concept study. In vitro experiments have defined the antiviral and resistance profile for GSK’254. The compound displayed strong antiviral activity against a library of subtype B and C chimeric viruses containing Gag polymorphisms and site-directed mutants previously shown to affect potency of earlier-generation MIs, with a mean protein-binding adjusted 90% effective concentration of 33 nM. Furthermore, GSK’254 exhibited robust antiviral activity against a panel of HIV-1 clinical isolates, with a mean EC 50 of 9 nM. Mechanistic studies established that bound GSK’254 dissociated on average 7.1-fold more slowly from wild-type Gag virus-like particles (VLPs) compared with a previous-generation MI. In resistance studies, the previously identified A364V Gag region mutation was selected under MI pressure in cell culture and during the phase IIa clinical study. As expected, GSK’254 inhibited cleavage of p25 in a range of polymorphic HIV-1 Gag VLPs. Virus-like particles containing the A364V mutation exhibited a p25 cleavage rate 9.3 times faster than wild-type, providing a possible mechanism for MI resistance. The findings demonstrate that GSK’254 potently inhibits a broad range of HIV-1 strains expressing Gag polymorphisms.

HIV-1 Gag Non-Cleavage Site PI Resistance Mutations Stabilize Protease/Gag Substrate Complexes in Silico via a Substrate-Clamp

HIV-1 protease active site inhibitors are a key part of antiretroviral therapy, though resistance can evolve rendering therapy ineffective. Protease inhibitor resistance typically starts with primary mutations around the active site, which reduces inhibitor binding, protease affinity for substrate cleavage site residues P4-P4′, and viral replication. This is often followed by secondary mutations in the protease substrate-grooves which restore viral replication by increasing protease affinity for cleavage site residues P12-P5/P5′-P12′, while maintaining resistance. However, mutations in Gag alone can also result in resistance. The Gag resistance mutations can occur in cleavage sites (P12-P12′) to increase PR binding, as well as at non-cleavage sites. Here we show in silico that Gag non-cleavage site protease inhibitor resistance mutations can stabilize protease binding to Gag cleavage sites which contain structured subdomains on both sides: SP1/NC, SP2/p6, and MA/CA. The Gag non-cleavage site resistance mutations coordinated a network of H-bond interactions between the adjacent structured subdomains of the Gag substrates to form a substrate-clamp around the protease bound to cleavage site residues P12-P12′. The substrate-clamp likely slows protease disassociation from the substrate, restoring the cleavage rate in the presence of the inhibitor. Native Gag substrates can also form somewhat weaker substrate-clamps. This explains the 350-fold slower cleavage rate for the Gag CA/SP1 cleavage site in that the CA-SP1 substrate lacks structured subdomains on both sides of the cleavage site, and so cannot form a substrate-clamp around the PR.

Stephan Oroszlan and the Proteolytic Processing of Retroviral Proteins: Following A Pro

Steve Oroszlan determined the sequences at the ends of virion proteins for a number of different retroviruses. This work led to the insight that the amino-terminal amino acid of the mature viral CA protein is always proline. In this remembrance, we review Steve’s work that led to this insight and show how that insight was a necessary precursor to the work we have done in the subsequent years exploring the cleavage rate determinants of viral protease processing sites and the multiple roles the amino-terminal proline of CA plays after protease cleavage liberates it from its position in a protease processing site.

Effect of follicular ablation and gonadotropin priming on the recovery and quality of oocytes in Boran cows

Genetic differences have been suggested as a possible cause for variation in responses to exogenous hormones. Here we evaluated the effect of follicle ablation, exogenous FSH and coasting time prior to ovum pick-up (OPU) on the number of follicles suitable for aspiration, oocyte quality, and cleavage rate in Ethiopian Boran cows. The experiment was carried out in three parts, I) Cows were synchronized using 500µg PGF2α given 11 days apart. Cows were then subjected to a biweekly ovum pickup session before ovulation (n=5) or starting Day 7 after ovulation (n=4) for three weeks. II) Cows were similarly synchronized and all visible follicles were ablated on the first days of overt estrus which were then further grouped into cows that received a divided dose of 350IU FSH (n=5) or 175IU FSH (n=5) over three days. In both groups OPU was carried out weekly starting 48h after the last FSH for six weeks. III) A similar protocol as in part II was carried out but coasting period was increased to 72hrs for cows that received 350IU FSH as divided dose (n=5) and 48hrs coasting period for single 350IU FSH dose (n=5). The covariates of follicles and oocyte were not affected (P>0.05) by corpus luteum presence at OPU. The mean number of medium (7.36±0.57) and large (8.28±0.96) follicles were significantly higher (P<0.05) in group that received divided 350IU FSH. Similarly, the mean number of Grade-1 (4.19±0.24) and Grade-2 (4.32±.27) COC, maturation rate (70.41%) and cleavage rate (47.5%) were significantly higher (P<0.05) in group that received 350IU FSH. COC quality was significantly (P<0.05) influenced by costing period. However, both maturation and cleavage rates were not affected by the coasting period. This study demonstrated that follicular ablation and treatment with FSH improves follicular population and oocyte recovery rate in Boran cows.

Effect of Administration of Single Dose GnRH Agonist in Luteal Phase on Implantation Rate of Fresh ICSI Cycles

The luteal phase (LP) in the fresh ICSI cycle is insufficient, adequate LP support is one of the approved treatments for improving implantation and pregnancy rates. It is generally known that the LP is inadequate after ovarian stimulation due to negative from supra-physiological blood levels of steroids released by numerous corporal luteal, LH concentrations are low during the luteal phase. In this study, patients were divided into two groups: (40) patients as study group; those who received GnRHa (Decapeptil 0.1 mg), three days after embryo transfer, in addition to conventional luteal phase support (LPS) in the LP to increase the implantation and pregnancy rate in IVF; and their control group (40) received standard LPS only. On the second day of stimulation, blood samples for FSH, LH, TSH, E2, and prolactin were taken. On the day of ovulation induction, measure E2, progesterone, and LH; and on the day of embryo transfers, measure progesterone and LH. The overall characteristics of the patients in both groups were not significantly different. There was also no significant change in the number of total oocytes, mean of metaphase II oocytes percent, cleavage rate, grade I embryo percent, or serum hormones level between the study and control groups (p > 0.05). GnRH agonist treatment in the luteal phase improves clinical pregnancy and implantation rate in fresh ICSI cycles but is not statistically significant.

PSV-5 Relationship between sire conception rate, sperm motility, sperm SERPINA5 relative concentration, and in vitro produced embryos in dairy bulls

Sire conception rate (SCR) is a field measure of fertility among bulls, but it can be influenced by several factors (Sperm transport, sperm-egg binding, early embryo development, etc). The objective of this study was to evaluate the relationship between SCR, sperm motility, SERPINA5 concentrations, and in vitro embryo development. Measurements were performed in 19 bulls with SCR values ranging from -7.7 to 4.45. For each bull, an aliquot of frozen-thawed semen was used for analyses of total (TMOT) and progressive (PROG) motility. Remaining semen was fixed with 2% formaldehyde, and concentration of SERPINA5 was determined by immunolocalization (antibody SERPINA5/Dylight405; PA5-79976-Invitrogen / ab201798-Abcam). Mean fluorescence intensity was determined in ~200 sperm heads/bull. Approximately 149 oocytes/bull were fertilized in vitro for embryo development analysis (cleavage and blastocyst rates). Statistical procedures were performed in SAS (9.4) using the procedures CORR for correlations (SCR, TMOT, PROG, SERPINA5, cleavage and blastocyst) and GLIMMIX for comparison of “field-fertility” (SCR divided in HIGH or LOW) and “field-embryo-fertility” (LOW-SCR sires were divided based on blastocyst rate (HIGH or LOW) resulting in two classifications; LOW-HIGH≥31% and LOW-LOW≤26%, respectively). There were positive correlations (P &lt; 0.05) between cleavage-blastocyst (r=0.50), SERPINA5-cleavage (r=0.48), and TMOT-PROG (r=0.76). Sire SCR was not associated with SERPINA5, TMOT, PROG, cleavage and blastocyst rate (P &gt; 0.52). Among LOW-SCR sires, LOW-LOW sires (-4.83±0.60) tended to have a better SCR score than LOW-HIGH (-6.18±0.42) sires (P = 0.08), but there were no differences (P &gt; 0.43) between LOW-HIGH, LOW-LOW, and HIGH sires for SERPINA5, TMOT, PROG, and cleavage. In conclusion, some LOW SCR sires have good embryo development indicating a different mechanism for their low SCR; however, these differences in SCR could not be explained by TMOT, PROG, SERPINA5, cleavage and blastocyst. There were, however, positive correlations between cleavage-blastocyst rate, and SERPINA5-cleavage rate.