1-Methyl-7-(4-nitro­phen­yl)-3-phenyl­pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine-6,8(3H,7H)-dione

In the title compound, C21H13N5O4, the dihedral angles formed between the planes of the phenyl and nitro­phenyl rings and that of the heterotricyclic plane are 41.29 (7) and 61.35 (6)°, respectively. In the crystal, weak C—H⋯O interactions help to establish the packing

Proteomic Analysis of Epididymal and Ejaculated Sperm and Respective Fluids

Study Description: Ejaculated and epididymal semen was collected from mature Angus bulls (n = 9), and then centrifuged to separate sperm and fluid. Fluids were collected and sperm pellets were resuspended in a high ionic solution and vortexed to remove loosely attached proteins. Sperm samples were centrifuged, and the supernatant was collected. Samples collected for protein analysis were snap frozen in liquid nitrogen and stored at -80 ºC. Protein analysis was performed by liquid chromatography with tandem mass spectrometry (LCMS/MS). Yearling Angus cross bulls (n = 40) were used for sperm cultures. Ejaculated (n = 20) and epididymal (n = 20) sperm were collected, diluted and cultured in a commercial media at pH 5.8, 6.8 and 7.3, at 4 ºC and evaluated for motility and viability every 24 h until motility was below 20%. There was an effect of pH, time and pH by time interaction for motility and viability for both ejaculated and epididymal sperm (P ≤ 0.05). At 216 h of incubation epididymal sperm at pH 7.3 and ejaculated sperm at pH 6.8 dropped below 20% motility. Overall, in all samples, a total of 458 unique proteins were identified. It was identified that ejaculated fluid and ejaculated sperm had 178 and 298 proteins, respectively. Also, it was identified that epididymal fluid and epididymal sperm had 311 and 344 proteins, respectively. There were Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways significantly enriched (FDR \u3c 0.05) for ejaculated fluid (n = 8), epididymal fluid (n = 24), ejaculated sperm (n = 10), and epididymal sperm (n = 18). The most important KEGG pathway identified was the metabolic pathway. Within the metabolic pathway the glycolysis/gluconeogenesis, pentose phosphate, and glutathione metabolism pathways were significantly enriched among proteins only present in epididymal samples. Other proteins identified that may be related to epididymal sperm’s increased longevity were peroxidases and glutathione peroxidases for their antioxidant properties

Proteomic analyses identify differences between bovine epididymal and ejaculated spermatozoa that contribute to longevity

Sperm are stored for extended periods of time in the epididymis, but upon ejaculation motility is increased and lifespan is decreased. The objective of this study was to identify differences in proteins between epididymis and ejaculated samples that are associated with longevity. Ejaculated semen was collected from mature Angus bulls (n = 9); bulls were slaughtered and epididymal semen was collected. Epididymal and ejaculated semen were centrifuged to separate sperm and fluid. Fluids were removed and sperm pellets were resuspended in a high ionic solution and vortexed to remove loosely attached proteins. Sperm samples were centrifuged, and the supernatant was removed; both fluid and sperm samples were snap frozen in liquid nitrogen and stored at -80 oC. Protein analysis was performed by LCMS/MS. A different group of yearling Angus cross bulls (n = 40) were used for sperm cultures. Ejaculated (n = 20) and epididymal (n = 20) semen were diluted and cultured in a commercial media at pH 5.8, 6.8 and 7.3, at 4 oC. Sperm were evaluated for motility and viability every 24 h until motility was lower than 20%. There was an effect of pH, time and pH by time interaction for motility and viability for both ejaculated and epididymal sperm (P ≤ 0.05). At 216 h of incubation epididymal sperm at pH 7.3 and ejaculated sperm at pH 6.8 reached motility below 20%. A total of 458 unique proteins were identified; 178, 298, 311, and 344 proteins were identified in ejaculated fluid, ejaculated sperm, epididymal fluid and epididymal sperm, respectively. There were 8, 24, 10, and 18 significant KEGG pathways (FDR \u3c0.05) for ejaculated fluid, epididymal fluid, ejaculated sperm, and epididymal sperm, respectively. The metabolic pathway was identified as the most important KEGG pathway; glycolysis/gluconeogenesis, pentose phosphate, and glutathione metabolism pathways were significant among proteins only present in epididymal samples within the metabolic pathway. Other proteins identified that may be related to epididymal sperm\u27s increased longevity were peroxidases and glutathione peroxidases for their antioxidant properties. In summary, energy metabolism in the epididymis appears to be more glycolytic compared to ejaculated and epididymis sperm have a larger number of antioxidants available which may be helping to maintain sperm in a quiescent state. Epididymal sperm remained viable (membrane integrity) longer than ejaculated sperm when cultured at the same pH

Use of Sperm Proteins as a Putative Fertility Marker

The objectives of this study were to characterize the variation and evaluate whether CD9 and SERPINA5 could be used as fertility markers in bovine sperm

Volume control of the lower limb with graduated compression during different muscle pump activation conditions and the relation to limb circumference variation

Background: The literature supports the use of graduated compression stockings (GCS) for leg edema. Nevertheless, there is a paucity of data on the GCS effect on limb edema related to sitting, standing, and walking. Data of different limb shapes and their impact on GCS-exerted pressure are lacking. This investigation provides evidence-based information on the effect of GCS on edema reduction and the impact of limb circumference gradients on GCS pressure. Methods: Thirty healthy individuals (15 men and 15 women; mean age, 32 ± 5 years) were included. All the participants underwent lower limb volume (Kuhnke formula) measurement, before and after sitting for 30 minutes, wearing below-ankle noncompressive socks. The same assessment was repeated 7 days later, in the same participants, but with wearing of below-knee 16 to 20 mm Hg GCS. At 7-day intervals, 1 week with below-ankle noncompressive socks and 1 week with below-knee 16 to 20 mm Hg GCS, all the participants repeated the same protocol including standing and walking. Ten participants underwent bioimpedance assessment (Biody Xpert II; eBIODY, La Ciotat, France) before and after sitting, standing, and walking. In the same group, B and B1 interface pressure values were measured. Results: Data collection was completed in all 60 limbs. Sitting or walking without GCS led to no significant volume changes, whereas volume was decreased by the use of GCS (−4.8% [P <.00001] and −4.4% [P <.00001], respectively). Standing up without GCS led to an increase in volume (2.7%; P <.0001), whereas limb volume was decreased (4.6%; P <.0001) by use of GCS. Bioimpedance showed extracellular water reduction only while walking with GCS (from 40.55% ± 1.66% to 40.45% ± 1.71%; P <.017). Mean interface pressure was 19 ± 5 mm Hg (B) and 16 ± 5 mm Hg (B1). The interface pressure variation from B to B1 was not homogeneous among participants (mean percentage variation of −13% ± 25%, ranging from −54% to 16%). A negative linear trend between pressure variation and circumference percentage increase was found; the subanalysis excluding the two outliers showed a strong negative linear correlation (Pearson coefficient r = −0.96). Conclusions: GCS led to a significant limb volume reduction irrespective of limb position and muscle pump function. However, extracellular fluid is mobilized only during muscle contraction while walking with GCS. Interestingly, different lower limb circumference variations influence the interface pressure gradient, indicating the importance of proper fitting of both B and B1 during prescription. These data provide a foundation to future investigations dealing with GCS effect on fluid mobilization and with limb geometry impact on compression performance

Purification of a monoclonal antibody using a novel high-capacity multimodal cation exchange nonwoven membrane

A high-capacity, multimodal cation exchange (MMC) chromatographic membrane was developed by conjugating a multimodal ligand – 2-mercaptopyridine-3-carboxylic acid (MPCA) – on a polybutylene terepthalate (PBT) nonwoven fabric. The membrane features an equilibrium binding capacity of ≈ 1000 mg of human polyclonal IgG (IgG) per g of membrane and dynamic binding capacities (DBC10%) ranging from 77.5 to 115.1 mg/mL (residence times of 1 and 5 min, respectively); these values are 2-to-3-fold higher than those of commercial MMC adsorbents. The effects of buffer composition, pH, conductivity on the binding behavior of the MMC-MPCA membrane were investigated in detail. As a moderate cation exchange binder, MPCA enables effective protein elution using buffers with mild pH (8.0–9.0) and conductivity (≈13 mS/cm), thus circumventing the harsh conditions often needed in multimodal chromatography. The MMC-MPCA membrane was evaluated for product capture in bind-and-elute mode on a Chinese hamster ovary (CHO) cell culture harvest containing therapeutic monoclonal antibodies, using commercial multimodal (Capto MMC and MX-Trp-650M) and affinity (AF-rProtein A HC-650F) resins as controls. The MMC-MPCA membrane outperformed the multimodal resins in terms of binding capacity as well as clearance of host cell proteins (HCPs) and aggregates. The membrane was then evaluated by polishing the mAb from a Protein A eluate in bind-and-elute mode. The MMC-MPCA membrane reduced the level of high molecular weight components from 11% to 4% and the HCP content from 1319.7 ppm to 48.7 ppm (LRV of 1.4). Most notably, proteomics analysis of the product demonstrated the clearance of a significant fraction of persistent, high-risk HCPs from the Protein A eluate

Thrombotic risk assessment in antiphospholipid syndrome: The role of new antibody specificities and thrombin generation assay

Antiphospholipid syndrome (APS) is an autoimmune condition characterized by the presence of antiphospholipid antibodies (aPL) in subjects presenting with thrombosis and/or pregnancy loss. The currently used classification criteria were updated in the international consensus held in Sidney in 2005. Vascular events seem to result of local procoagulative alterations upon triggers influence (the so called “second-hit theory”), while placental thrombosis and complement activation seem to lead to pregnancy morbidity. The laboratory tests suggested by the current classification criteria include lupus anticoagulant, a functional coagulation assay, and anticardiolipin and anti-β2-glycoprotein-I antibodies, generally detected by solid phase enzyme-linked immunosorbent assay. The real challenge for treating physicians is understanding what is the actual weight of aPL in provoking clinical manifestations in each case. As thrombosis has a multi-factorial cause, each patient needs a risk-stratified approach. In this review we discuss the role of thrombotic risk assessment in primary and secondary prevention of venous and arterial thromboembolic disease in patients with APS, focusing on new antibody specificities, available risk scoring models and new coagulation assays

Use of Pregnancy Associated Glycoproteins to determine Fetal Age Throughout Gestation

The objective of the current study was to determine if a commercially available blood pregnancy test could be modified to detect differences in pregnancy-associated glycoprotein (PAG) concentrations that would be indicative of stage of pregnancy or fetal age