Intact Plasma Research Articles

Differences in the properties of porcine cortical and nuclear fiber cell plasma membranes revealed by saturation recovery EPR spin labeling measurements

Eye lens membranes are complex biological samples. They consist of a variety of lipids that form the lipid bilayer matrix, integral proteins embedded into the lipid bilayer, and peripheral proteins. This molecular diversity in membrane composition induces formation of lipid domains with particular physical properties that are responsible for the maintenance of proper membrane functions. These domains can be, and have been, effectively described in terms of the rotational diffusion of lipid spin labels and oxygen collision with spin labels using the saturation recovery (SR) electron paramagnetic resonance method and, now, using stretched exponential function for the analysis of SR signals. Here, we report the application of the stretched exponential function analysis of SR electron paramagnetic resonance signals coming from cholesterol analog, androstane spin label (ASL) in the lipid bilayer portion of intact fiber cell plasma membranes (IMs) isolated from the cortex and nucleus of porcine eye lenses. Further, we compare the properties of these IMs with model lens lipid membranes (LLMs) derived from the total lipids extracted from cortical and nuclear IMs. With this approach, the IM can be characterized by the continuous probability density distribution of the spin-lattice relaxation rates associated with the rotational diffusion of a spin label, and by the distribution of the oxygen transport parameter within the IM (i.e., the collision rate of molecular oxygen with the spin label). We found that the cortical and nuclear LLMs possess very different, albeit homogenous, spin lattice relaxation rates due to the rotational diffusion of ASL, indicating that the local rigidity around the spin label in nuclear LLMs is considerably greater than that in cortical LLMs. However, the oxygen transport parameter around the spin label is very similar and slightly heterogenous for LLMs from both sources. This heterogeneity was previously missed when distinct exponential analysis was used. The spin lattice relaxation rates due to either the rotational diffusion of ASL or the oxygen collision with the spin label in nuclear IMs have slower values and wider distributions compared with those of cortical IMs. From this evidence, we conclude that lipids in nuclear IMs are less fluid and more heterogeneous than those in cortical membranes. Additionally, a comparison of properties of IMs with corresponding LLMs, and lipid and protein composition analysis, allow us to conclude that the decreased lipid-to-protein ratio not only induces greater rigidity of nuclear IMs, but also creates domains with the considerably decreased and variable oxygen accessibility. The advantages and disadvantages of this method, as well as its use for the cluster analysis, are discussed.

Green tea extract increases the quality and reduced DNA mutation of post-thawed Kacang buck sperm

The study aimed to determine the addition of green tea extract (GTE) in extender on the quality and DNA mutation of post-thawed Kacang buck sperm. The sperm DNA mutation was observed on nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase 1 (ND1) of mitochondrial Deoxyribonucleic Acid (mtDNA). A pool of 12 Kacang buck ejaculates was diluted in skim milk-egg yolk extender contained 0, 0.05, 0.10, and 0.15 mg of GTE/100 mL for T0, T1, T2, and T3 group, respectively. Each of the aliquot groups was packaged in 0.25 mL French mini straw contained 60 million alive sperm and froze according to the protocol. The ND1 mtDNA amplification of samples was carried out Polymerase Chain Reaction machine, followed by DNA sequencing using the Sanger method. Meanwhile, the phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA 7.0 software. The results showed that the T2 group maintained the highest quality for Kacang buck post-thawed semen. There was the highest percentages of sperms viability, motility, intact plasma membrane (IPM), the lowest of malondialdehyde (MDA) concentration, sperm DNA fragmentation (SDF), the total and types of ND1 mtDNA mutation frequency. The phylogenetic tree analysis revealed that the clade of the T2 group was most closely related to the sequence reference. However, there was no correlation between the semen quality parameters (sperm viability, motility, IPM, MDA concentration, and SDF) with ND1 mtDNA mutation of post-thawed Kacang buck semen. It could be concluded that GTE was useful as an antioxidant for Kacang buck semen extender for frozen sperm.

Drug resistance mutations in HIV provirus are associated with defective proviral genomes with hypermutation.

HIV proviral sequencing overcomes the limit of plasma viral load requirement by detecting all the 'archived mutations', but the clinical relevance remains to be evaluated. We included 25 participants with available proviral sequences (both intact and defective sequences available) and utilized the genotypic sensitivity score (GSS) to evaluate the level of resistance in their provirus and plasma virus. Defective sequences were further categorized as sequences with and without hypermutations. Personalized GSS score and total GSS score were calculated to evaluate the level of resistance to a whole panel of antiretroviral therapies and to certain antiretroviral therapy that a participant was using. The rate of sequences with drug resistance mutations (DRMs) within each sequence compartment (intact, defective and plasma viral sequences) was calculated for each participant. Defective proviral sequences harbored more DRMs than other sequence compartments, with a median DRM rate of 0.25 compared with intact sequences (0.0, P = 0.014) and plasma sequences (0.095, P = 0.30). Defective sequences with hypermutations were the major source of DRMs, with a median DRM rate of 1.0 compared with defective sequences without hypermutations (0.042, P < 0.001). Certain Apolipoprotein B Editing Complex 3-related DRMs including reverse transcriptase gene mutations M184I, E138K, M230I, G190E and protease gene mutations M46I, D30N were enriched in hypermutated sequences but not in intact sequences or plasma sequences. All the hypermutated sequences had premature stop codons due to Apolipoprotein B Editing Complex 3. Proviral sequencing may overestimate DRMs as a result of hypermutations. Removing hypermutated sequences is essential in the interpretation of proviral drug resistance testing.

Protective effects of hesperetin on the quality of sperm, apoptosis, lipid peroxidation, and oxidative stress during the process of cryopreservation: An experimental study.

BackgroundHesperetin is a bioflavonoid compound, largely used in Chinese traditional medicine and found plenty in citrus fruits. Hesperetin has beneficial effects against different diseases. The sperm cryopreservation process is a common method that is used in infertility laboratories. It has been reported that during the cryopreservation process, the quality of sperm is significantly reduced.ObjectiveTo investigate the effect of hesperetin on the quality of human spermatozoa during the cryopreservation process.Materials and Methods In this experimental study, 22 sperm sample of normozoospermia men who reffered to the infertility department of the Shariati Hospital (Tehran, Iran) Between October and November 2019 were collect and divided in to three groups as: 1) fresh, 2) control (frozen-thawed group without treatment), and 3) treatment group as frozen-thawed samples supplemented with 20 µM hesperetin. Motility, Viability, morphology, Apoptotic-like changes, intracellular HO, intracellular O2, and lipid peroxidation (LPO) was measured.Results Hesperetin treatment during the cryopreservation process of human sperm significantly improved the viability, motility, and morphology rates of the spermatozoa after frozen-thawed process in control group (p 0.01). In addition, it significantly reduced the reactive oxygen species (ROS) level, LPO level and increased the percentage of viable sperm cells with intact plasma membrane (p 0.01) after frozen-thawed process.ConclusionHesperetin can improve the quality of human sperm and also protect human sperm against reactive oxygen species, LPO, and apoptosis during the cryopreservation-thawing process.

Semen quality of Colombian Creole as compared to commercial pig breeds.

Characterization of Creole breeds is still very limited, including reproductive performance. In this research, we assessed the semen quality of three Colombian Creole breeds (Zungo, Casco de Mula and San Pedreño) relative to that of international breeds (Duroc, Belgian Landrace and Pietrain). Two doses from seven boars per breed were evaluated for sperm kinetics and membrane and acrosome integrity using computer-assisted sperm analysis (CASA) and flow cytometry, respectively. The Creole pigs showed lower (P < 0.05) volume of fluid ejaculated (185.5mL vs 239.9mL), and sperm concentration (340.5 × 106 vs 395.4 × 106 sperm/mL), motility (90.9% vs 95.3%) and progressive motility (63.1% vs 67.2%) than international breeds. No relevant differences between Creole and international breeds for sperm velocity traits were observed, but Creole boars had lower (P < 0.05) proportion of morphologic normal sperm (86.1% vs 90.6%) and of sperm with both intact plasma membrane and acrosome integrity (76.8% vs 87.5%). Mitochondrial membrane potential did not differ between breeds. Creole breeds in general produced less normal and motile sperm per ejaculate than international breeds (49.3 × 109 vs 81.5 × 109). Although San Pedreño had larger ejaculates than Zungo and Zungo had a greater proportion of normal and motile sperm than San Pedreño, Creole breeds did not differ in total amount of normal and motile sperm per ejaculate. The semen from Colombian Creole pigs is qualitatively acceptable, being less abundant but rich in normal and motile sperm, than that from commercial breeds. This should be considered when developing recommendations for semen use and conservation for AI in Creole pigs.

Effects of resveratrol in bull semen extender on post-thaw sperm quality and capacity for fertilization and embryo development

Resveratrol, a potent antioxidant, can be an alternative semen extender constituent to protect spermatozoa against reactive oxygen species (ROS); however, effects on sperm quality post-thawing and sperm function is not well understood. This study, therefore, was conducted to investigate effects of resveratrol supplementation to semen extender on sperm quality post-thawing. Bull semen was cryopreserved using extenders not supplemented or supplemented with 0.05, 0.1, or 1 mM resveratrol. Supplementation of extender with resveratrol at 0.05 mM resulted in greater (P < 0.05) sperm progressive motility, average path velocity, straight linear velocity, linearity and straightness when compared with no or 1 mM supplementations. Furthermore, effects of 0.05 mM resveratrol supplementations on plasma membrane and acrosome integrity and sperm fertilization capacity using in vitro procedures were investigated. Supplementation of semen extender with resveratrol resulted in a greater (P < 0.05) proportion of frozen-thawed spermatozoa with an intact acrosome and plasma membrane. Results from in vitro fertilization studies indicated there were no differences (P> 0.05) when there was no supplementation or supplementation with 0.05 mM resveratrol on embryo development to the cleavage and blastocyst stages. In conclusion, addition of resveratrol to bull semen extender resulted in greater sperm quality post-thawing in a dose-dependent manner, with values for variables related to sperm quality being greater when there was resveratrol supplementation at the 0.05 mM concentration. Proportion of embryo developing to the cleavage and blastocyst stages after in vitro fertilization was not affected by resveratrol supplementation to semen extenders.

TGIRT-seq Protocol for the Comprehensive Profiling of Coding and Non-coding RNA Biotypes in Cellular, Extracellular Vesicle, and Plasma RNAs.

High-throughput RNA sequencing (RNA-seq) has extraordinarily advanced our understanding of gene expression and disease etiology, and is a powerful tool for the identification of biomarkers in a wide range of organisms. However, most RNA-seq methods rely on retroviral reverse transcriptases (RTs), enzymes that have inherently low fidelity and processivity, to convert RNAs into cDNAs for sequencing. Here, we describe an RNA-seq protocol using Thermostable Group II Intron Reverse Transcriptases (TGIRTs), which have high fidelity, processivity, and strand-displacement activity, as well as a proficient template-switching activity that enables efficient and seamless RNA-seq adapter addition. By combining these activities, TGIRT-seq enables the simultaneous profiling of all RNA biotypes from small amounts of starting material, with superior RNA-seq metrics, and unprecedented ability to sequence structured RNAs. The TGIRT-seq protocol for Illumina sequencing consists of three steps: (i) addition of a 3' RNA-seq adapter, coupled to the initiation of cDNA synthesis at the 3' end of a target RNA, via template switching from a synthetic adapter RNA/DNA starter duplex; (ii) addition of a 5' RNA-seq adapter, by using thermostable 5' App DNA/RNA ligase to ligate an adapter oligonucleotide to the 3' end of the completed cDNA; (iii) minimal PCR amplification, to add capture sites and indices for Illumina sequencing. TGIRT-seq for the Illumina sequencing platform has been used for comprehensive profiling of coding and non-coding RNAs in ribodepleted, chemically fragmented cellular RNAs, and for the analysis of intact (non-chemically fragmented) cellular, extracellular vesicle (EV), and plasma RNAs, where it yields continuous full-length end-to-end sequences of structured small non-coding RNAs (sncRNAs), including tRNAs, snoRNAs, snRNAs, pre-miRNAs, and full-length excised linear intron (FLEXI) RNAs. Graphic abstract: Figure 1.Overview of the TGIRT-seq protocol for Illumina sequencing.Major steps are: (1) Template switching from a synthetic R2 RNA/R2R DNA starter duplex with a 1-nt 3' DNA overhang (a mixture of A, C, G, and T residues, denoted N) that base pairs to the 3' nucleotide of a target RNA, and upon initiating reverse transcription by adding dNTPs, seamlessly links an R2R adapter to the 5' end of the resulting cDNA; (2) Ligation of an R1R adapter to the 3' end of the completed cDNA; and (3) Minimal PCR amplification with primers that add Illumina capture sites (P5 and P7) and barcode sequences (indices 5 and 7). The index 7 barcode is required, while the index 5 barcode is optional, to provide unique dual indices (UDIs).

Ultrastructural analysis of Lyophilized Human Spermatozoa.

Objective:Lyophilization is potentially more practical and cost-effective alternative for sperm preservation. However, there are no studies that evaluate the ultrastructure of human spermatozoa after lyophilization. Therefore, the aim of our study was to evaluate the ultrasctructure of lyophilized spermatozoa using Transmission Electron Microscopy.Methods:From a total of 21 donated seminal samples, 30 aliquots were originated and divided into two aliquots so that one could have been submitted to cryopreservation/thaw and the other for lyophilization/rehydration. The liquefied aliquots were homogenized at room temperature. Samples assigned for cryopreservation were placed in straws and samples assigned for lyophilization were placed in the appropriate vials. Cryopreservation samples were placed at -30oC for 30 minutes subsequently for 30 minutes at vapour phase and then plunged into liquid nitrogen. Lately, were warmed in water bath at 37oC for 10 minutes followed by 10 minutes centrifugation. The pellet was resuspended and analysed in a Makler chamber. The semen vials assigned for lyophilization were loaded into a pre-fixed freeze-drying chamber. Following lyophilization, vials were removed from the freeze-drying chamber and kept at 4oC until rehydration. TEM was performed after rehydration and thawing. Sperm samples were fixed, rinsed in buffer, post fixed and dehydration was carried out in escalating concentrations of alcohol solution, acetone and then, embedding in Epon resin. Ultrathin sections were stained and examined in a Transmission Electron Microscope.Results:Analysis of sperm after freezing/thawing using Transmission Electron Microscopy showed lesions to the midpiece, with some mitochondria degeneration and random rupture of plasma membrane. In the head, we identified intact plasma membrane, nucleus and acrosome, as in the flagellum all main structures remained intact including the plasma membrane, the longitudinal columns of dense fibers and the semicircular fibers. Analysis by Transmission Electron Microscopy showed that spermatozoa heads had ruptured plasma membranes, absence of acrosomes, nuclei with heterogeneous and decompressed chromatin. Mitochondria were deteriorated in the midpiece. Longitudinal columns of dense fibers were absent in the flagellum. Axonemes, in cross-sections, were disrupted with disorganized structures.Conclusions:To our knowledge, our study demonstrated, for the first time, the structure of the human spermatozoa after lyophilization using Transmission Electron Microscopy. The use of a fixed lyophilization protocol with media containing cryoprotectants might explain the damage to the structures. More studies are necessary to improve the results of sperm lyophilization. In the future, the use of lyophilization of spermatozoa might reduce the costs of fertility preservation, since there will be no need for storage space and transportation is simpler.

Resveratrol supplementation into extender protects against cryodamage in dog post-thaw sperm

Antioxidants have multiple protective roles in a variety of cells and thus can be used to protect sperm against cryo-damage during freezing, which affects fertility. The antioxidant resveratrol (3,5,4-trihydroxytrans-stilbene; RSV) has been reported to protect the animal sperm during cryopreservation, including human sperm. In this study, we assessed the protective effects of RSV supplementation on dog sperm cryopreservation. Semen was collected from four dogs and the effect of different concentrations of RSV (0, 100, 200, and 400 µM) on post-thaw sperm quality was examined. After thawing, sperm motility was assessed using computer-aided sperm analysis, and the structural integrity of the plasma membrane, acrosome, and chromatin was examined. In addition, their mitochondrial activity and gene expression were also assessed. Dog sperm cryopreserved with 200 µM RSV showed significant improvement in post-thaw sperm motility and viability compared with that of the control group (P<0.05). Moreover, RSV-supplemented samples showed significantly higher numbers of sperm with an intact plasma membrane, active mitochondria, and structural integrity of acrosomes and chromatin than that of control samples (P<0.05). Furthermore, gene expression showed that RSV supplemented samples showed lower expression of pro-apoptotic (BAX), reactive oxygen species (ROS) modulator oxidative stress-related (ROMO1) and 8-oxoguanine DNA glycosylase 1 (OGG1) whereas higher expression levels of anti‐apoptotic (BCL2), protamine-2 (PRM2), protamine-3 (PRM3) and sperm acrosome‐associated 3 (SPACA3) genes than control. Our results suggest that RSV, at its optimum concentration, can be efficiently used as an antioxidant in the cryopreservation of dog sperm.

Super-Resolution Imaging and Spatial Analysis of RAS on Intact Plasma Membrane Sheets.

The function of lipid-anchored small GTPases RAS proteins is mostly compartmentalized to the plasma membrane (PM). Complex biophysical interactions between the C-terminal membrane-anchoring domains of RAS isoforms and PM lipids drive spatial segregation of RAS molecules in the formation of nanometer-sized domains, termed as nanoclusters. These RAS/lipid proteolipid nano-assemblies are the main sites for efficient effector recruitment and signal transduction. Here, we describe a super-resolution imaging method to quantify the nanometer-sized nanoclustering of RAS over a length scale between 8 and 240nm on intact PM sheets of mammalian cells. Detailed molecular spatial distribution parameters, including the extent of nanoclustering, average cluster size, clustered fraction, and population distribution can be obtained by the univariate spatial distribution analysis. Intermolecular associations between different RAS isoforms, RAS and various PM lipids, as well as RAS and diverse effectors can be quantified via bivariate co-localization analysis.

A Technology for Anti-Thrombogenic Drug Coating of Small-Diameter Biodegradable Vascular Prostheses.

The aim of the study was to develop a technology for anti-thrombogenic drug coating of biodegradable porous scaffolds and to evaluate the physicomechanical and hemocompatible properties of functionally active vascular prostheses with and without a drug coating.Materials and Methods.Vascular prostheses from polyhydroxybutyrate/valerate and polycaprolactone with the incorporated vascular endothelial growth factor, the main fibroblast growth factor, and the chemoattractant SDF-1α were made by emulsion electrospinning. Additional surface modification of the prostheses was carried out by forming a hydrogel coating of polyvinylpyrrolidone capable of binding drugs as a result of complexation. Unfractionated heparin and iloprost were used as anti-thrombogenic drugs.Results.We show that after the modification of vascular prostheses with heparin and iloprost, a 5.8-fold increase in the Young’s modulus value was noted, which indicated a greater stiffness of these grafts compared to the unmodified controls. Platelet aggregation on the surface of heparin + iloprost coated vascular prostheses was 3.3 times less than that with the unmodified controls, and 1.8 times less compared to intact platelet-rich plasma. The surface of vascular prostheses with heparin and iloprost was resistant to adhesion of platelets and blood proteins.Conclusion.Drug (unfractionated heparin and iloprost) coating of the surface of biodegradable prostheses significantly improved the anti-thrombogenic properties of these grafts but contributed to the increased stiffness of the prostheses.

Effect of sulforaphane on apoptosis, reactive oxygen species and lipids peroxidation of human sperm during cryopreservation

Sperm cryopreservation is a common procedure to preserve viable sperm for an indefinite period. This procedure has numerous detrimental effects on sperm function due to increased generation of reactive oxygen species (ROS). During cryopreservation, while ROS increases, antioxidant enzymes level decreases. It has been shown that a relationship exist between lower antioxidant levels and infertility. l-Sulforaphane (SFN) is an isothiocyanate in cruciferous vegetables of the brassica class that has potent protective effects against oxidative stress. The purpose of the present study was to evaluate the effects of SFN supplementation during the freeze-thaw process on different parameters of human spermatozoa which can influence sperm fertilizing ability. Samples were collected from 25 healthy men and each sample was divided into three groups: fresh, control (untreated frozen/thawed samples) and treatment (treated frozen/thawed with SFN) groups. Sperm parameters, ROS production (using flow cytometry), plasma membrane integrity (using flow cytometry), Lipid peroxidation (using ELISA) were evaluated. Our results demonstrated that 5 μM SFN improved all parameters of sperm including viability (P < 0.001), motility, and morphology (P < 0.05) after the freeze-thaw process. Furthermore, SFN reduced the levels of intracellular hydrogen peroxide (P < 0.01) and superoxide anion (P < 0.05). Also, SFN significantly increased the percentage of viable sperm cells with the intact plasma membrane (P < 0.001) and decreased the level of lipid peroxidation after the freeze-thaw process (P < 0.01).Our findings showed that spermatozoa treatment with 5 μM SFN before the freeze-thaw process has protective effects against oxidative stress and could decrease the detrimental effects of this process on sperm quality.

Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils.

Simple SummaryDonkey Artificial Insemination (AI) with frozen/thawed semen results in poor fertility outcomes. Jennies show a significant post-AI endometrial reaction, with a large amount of defense cells—polymorphonuclear neutrophils (PMN)—migrating to the uterine lumen. Seminal plasma (SP) has a detrimental effect on sperm conservation and its removal is a necessary step in the semen freezing protocol. However, several SP proteins seem to control sperm-PMN binding. Single layer centrifugation (SLC) with colloids, which has been used to select spermatozoa and improve reproductive performance in different species, is known to remove SP proteins attached to the sperm membrane. In this study, two experiments were performed. The first one compared the quality of SLC-selected and non-selected fresh donkey spermatozoa. In the second experiment, PMN obtained from the peripheral blood were co-incubated with selected and unselected spermatozoa, and the interaction between PMN and spermatozoa was analyzed. In conclusion, SLC of fresh donkey semen increases the proportion of functionally intact spermatozoa and appears to remove the SP proteins that inhibit sperm-PMN binding, thus increasing sperm phagocytosis by PMN.This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 °C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding.

Effect of Simmental bull seminal plasma protein in egg yolk-citrate extender on Kacang buck semen fertility

The genetic resources of Indonesia's indigenous Kacang goat require preservation. Artificial insemination is expected to accelerate population increases and preserve genetic resources simultaneously. The present study was the maiden attempt for cryopreservation of Kacang buck sperm. The objectives of this study were to determine whether the supplementation of superior Simmental bull seminal plasma protein in egg yolk-citrate extender could improve the quality of post-thawed Kacang buck sperm, increase conceptions rates, and improve kidding rates. Buck semen was diluted without supplementation (T0) and with supplementation of 2.5 mg (T1) and 5 mg (T2) of Simmental bull seminal plasma protein per mL egg yolk-citrate extender. Extended semen was packed in 0.25 mL straw as cryopreserved frozen semen. Post-thawed semen samples were evaluated for viability, motility, intact plasma membranes, malondialdehyde level, and DNA fragmentation. Estrus was synchronized for sixty Kacang does, which were divided randomly into three groups and inseminated using post-thawed semen. The progesterone serum concentration of the does was measured 7 and 22 days post-insemination to detect ovulation and conception. Pregnancy was confirmed using abdominal palpation at 43 days post-insemination and by observing birth. The T1 group showed the highest (P < 0.05) post-thawed viability, motility, and intact plasma membrane. Conception, pregnancy and kidding rates were also higher in T1 than other treatment groups. In conclusion, the 2.5 mg Simmental bull seminal plasma protein supplementation per mL egg yolk-citrate extender provided the best seminal quality and fertility of post-thawed Kacang buck semen.

Effect of addition an amino acid or its combination with EDTA on DNA integrity and morphometry sperm heads of freeze-dried bovine spermatozoa

The objective of the current study was to investigate the effect of addition an amino acid or its combination with EDTA on DNA integrity and morphometry sperm heads of freeze-dried bovine spermatozoa. Spermatozoa were freeze-dried in medium with the addition of an amino acid glycine, cysteine, glutamine, or its combination with EDTA. Sperm head morphometry was identified at 400X magnification using Axio Vision(Zeiss Company, Germany), while for membrane plasma integrity (MPI) determined by calculation of the percentage of spermatozoa having intact plasma membrane by osmotic resistance test method and DNA integrity analysis using acridine orange staining. Sperm head had declined in size after the freeze-drying process, MPI of FD spermatozoa gradually increased significantly when FD solution was added with an amino acid solution (glycine, cysteine) and its combination with EDTA. DNA integrity of all freeze-dried spermatozoa treatments was remaining intact, no significantly different (P&gt;0.01) among treatments. The present study concluded that the addition of an amino acid (glycine, cysteine) or its combination with EDTA could be reduced morphometric sperm heads and plasma membrane damage of freeze-dried bovine spermatozoa, however, DNA integrity of bovine sperm nucleus remaining intact after the freeze-drying process without addition both amino acids and EDTA.

Genetic pathways disrupted by ENPP1 deficiency provide insight into mechanisms of osteoporosis, osteomalacia, and paradoxical mineralization.

Ectonucleotide phosphatase/phosphodiesterase 1 (ENPP1) deficiency results in either lethal arterial calcifications ('Generalized Arterial Calcification of Infancy' - GACI), phosphate wasting rickets ('Autosomal Recessive Hypophosphatemic Rickets type 2' - ARHR2), early onset osteoporosis, or progressive spinal rigidity ('Ossification of the Posterior Longitudinal Ligament' - OPLL). As ENPP1 generates a strong endogenous mineralization inhibitor - extracellular pyrophosphate (PPi) - ENPP1 deficiency should not result in reduced bone volume, and therefore the mechanism ENPP1 associated osteoporosis is not apparent given current understanding of the enzyme's function. To investigate genetic pathways driving the skeletal phenotype of ENPP1 deficiency we compared gene expression in Enpp1asj/asj mice and WT sibling pairs by RNAseq and qPCR in whole bones, and in the liver and kidney by qPCR, directly correlating gene expression with measures of bone microarchitectural and biomechanical phenotypes. Unbiased analysis of the differentially expressed genes compared to relevant human disease phenotypes revealed that Enpp1asj/asj mice exhibit strong signatures of osteoporosis, ARHR2 and OPLL. We found that ENPP1 deficient mice exhibited reduced gene transcription of Wnt ligands in whole bone and increased transcription of soluble Wnt inhibitors in the liver and kidney, suggestive of multiorgan inhibition of Wnt activity. Consistent with Wnt suppression in bone, Collagen gene pathways in bone were significantly decreased and Fgf23 was significantly increased, all of which directly correlated with bone microarchitectural defects and fracture risk in Enpp1asj/asj mice. Moreover, the bone findings in 10-week old mice correlated with Enpp1 transcript counts but not plasma [PPi], suggesting that the skeletal phenotype at 10weeks is driven by catalytically independent ENPP1 function. In contrast, the bone findings in 23-week Enpp1asj/asj mice strongly correlated with plasma PPi, suggesting that chronically low PPi drives the skeletal phenotype in older mice. Finally, correlation between Enpp1 and Fgf23 transcription suggested ENPP1 regulation of Fgf23, which we confirmed by dosing Enpp1asj/asj mice with soluble ENPP1-Fc and observing suppression of intact plasma FGF23 and ALP. In summary, our findings suggest that osteoporosis associated with ENPP1 deficiency involves the suppression of Wnt via catalytically independent Enpp1 pathways, and validates Enpp1asj/asj mice as tools to better understand OPLL and Paradoxical Mineralization Disorders.

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy.

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Revisiting the secretion mechanism(s) of macrophage migration inhibitory factor-welcome to the "UPS club".

Revisiting the secretion mechanism(s) of macrophage migration inhibitory factor-welcome to the "UPS club".

Oxygen Transport Parameter in Plasma Membrane of Eye Lens Fiber Cells by Saturation Recovery EPR.

A probability distribution of rate constants contained within an exponential-like saturation recovery (SR) electron paramagnetic resonance signal can be constructed using stretched exponential function fitting parameters. Previously (Stein et al. Appl. Magn. Reson. 2019.), application of this method was limited to the case where only one relaxation process, namely spin-lattice relaxations due to the rotational diffusion of the spin labels in the intact eye-lens membranes, contributed to an exponential-like SR signal. These conditions were achieved for thoroughly deoxygenated samples. Here, the case is described where the second relaxation process, namely Heisenberg exchange between the spin label and molecular oxygen that occurs during bimolecular collisions, contributes to the decay of SR signals. We have further developed the theory for application of stretched exponential function to analyze SR signals involving these two processes. This new approach allows separation of stretched exponential parameters, namely characteristic stretched rates and heterogeneity parameters for both processes. Knowing these parameters allowed us to separately construct the probability distributions of spin-lattice relaxation rates determined by the rotational diffusion of spin labels and the distribution of relaxations induced strictly by collisions with molecular oxygen. The later distribution is determined by the distribution of oxygen diffusion concentration products within the membrane, which forms a sensitive new way to describe membrane fluidity and heterogeneity. This method was validated in silico and by fitting SR signals from spin-labeled intact nuclear fiber cell plasma membranes extracted from porcine eye lenses equilibrated with different fractions of air.

PKPD Modeling and Dosing Considerations in Advanced Ovarian Cancer Patients Treated with Cisplatin-Based Intraoperative Intraperitoneal Chemotherapy

Intraperitoneal chemoperfusion (IPEC) of cisplatin is a popular treatment for advanced ovarian cancer, typically under hyperthermia (HIPEC). The use of cisplatin under (H)IPEC is off-label, and the role of hyperthermia is unknown. The aim of this study was to characterize the pharmacokinetic/pharmacodynamic (PKPD) properties of cisplatin under (H)IPEC and to predict the optimal treatment regimen. Using a randomized design, data on intact cisplatin perfusate and plasma concentrations, leukocyte counts-a hematotoxicity marker-and serum creatinine-a nephrotoxicity marker-were collected from 50 patients treated with a combination of cytoreductive surgery (CRS) and either normothermic or hyperthermic IPEC of cisplatin dosed at 75, 100, and 120mg/m2. The non-linear mixed effects modeling technique was used to construct the PKPD models. The PK of intact cisplatin was characterized by a two-compartment model. A semi-physiological myelosuppression model for the leukopenia was modified to account for the CRS-induced leukocytosis and the residual myelosuppression effect of neoadjuvant chemotherapy. The incidence and severity of nephrotoxicity were described by a discrete-time Markov model. Hyperthermia increased the absorption rate of cisplatin by 16.3% but did not show a clinically relevant impact on the investigated toxicities compared with normothermia. Leukopenia was not severe, but nephrotoxicity can become severe or life-threatening and was affected by the dose and IPEC duration. The model predicted that nephrotoxicity is minimal at a cisplatin dose of 75mg/m2 with an IPEC duration of 1-2h and an 1-h duration is favored for doses between 100 and 120mg/m2. Graphical abstract.