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Heparin-induced thrombocytopenia (HIT) is a serious complication in patients exposed to heparin, leading to thrombocytopenia and, potentially, thrombosis. This disorder is challenging in cardiac surgery when anticoagulation for cardiopulmonary bypass is required. Herein a patient with HIT who had active thrombosis and successfully underwent urgent left ventricular assist device implantation managed with plasma exchange, intravenous immunoglobulin, and protamine infusion is described. These therapies reduce the immune response to heparin and minimize thrombosis when heparin reexposure is planned. These approaches to perioperative management of HIT represent an attractive alternative to the use of non-heparin anticoagulants in the cardiac and vascular surgical population.

Recurrent observation of clot in the cardiopulmonary bypass circuit after the administration of a protamine test dose (PTD) prompted concern over the effects of PTDs on patient activated clotting times (ACTs).Data were prospectively collected on 120 patients who had cardiopulmonary bypass while undergoing a variety of cardiac surgical procedures from July to October 2018 at the Toronto General Hospital, Toronto, Canada. ACTs were documented before cardiopulmonary bypass termination, after PTDs, and after protamine full doses. Statistical analysis was completed using a paired t test.The average PTD was calculated to be 36 ± 21 mg or 11% ± 7% of the full protamine dose of 367 ± 153 mg. This "test" dose ranged from 1% to 67% of full dose depending on the anesthetist. Post-PTD ACTs were widely variable. On average, there was a 40% ± 25% drop from the last ACT during cardiopulmonary bypass (650 ± 155 seconds) to the ACT after PTD (376 ± 153 seconds) (P.0001). In fact, 81% ± 5% of the patients' post-PTD ACTs were lower than the institutional ACT standard of 480 seconds for safe cardiopulmonary bypass initiation.Regardless of the PTD, there is no reliable way to predict how a patient's ACT will respond to a PTD. Clot formation is possible and circuit integrity is at risk when pump suction devices are continuously in use during PTD administration. Therefore, the study investigators strongly recommend that the direct recovery of mediastinal shed blood into the pump circuit be discontinued before any amount of protamine is administered to the patient.

Protamine is now well recognized as a key heparin neutralizing agent. However, protamine was discovered over a century ago, during experiments performed to uncover the secrets behind heritability. Although protamine was discovered as a highly charged protein, it did not receive the attention it deserved until the dawn of insulin era, when it was used to create the neutral protamine Hagedorn formulation. Based on the same principles, protamine was identified to neutralize heparin and has since been used successfully for many years in cardiothoracic surgery. More recently, its clinical applications have extended to gene therapy. In this historical sketch, the journey from the discovery of protamine, onwards to heparin neutralization, and up to its utilization in genetic modulatory treatments is detailed.

Nano drug delivery has been recently used to enhance the stability and bioavailability of chemotherapeutic agents. In this study, Chitosan/protamine nanocarrier was synthesized and used to encapsulate curcumin (CUR). The physicochemical properties of the empty carrier (CHPNPs) and curcumin-containing carrier (CU-CHPNPs) were characterized by TEM imaging, Zetasizer, and FT-IR spectroscopy. The antitumor activity of the prepared nanoparticles was assessed by determination of cell count, cell viability, the level of NF-κB, IL-6, and TNF-α and Bcl-2 gene expression in breast cancer cells (MCF-7). The results revealed that the obtained CU-CHPNPs had an average hydrodynamic size of 200 nm, zeta potential of +26.66 mv, and showed a drug encapsulation efficiency of 67%, and drug loading capacity of 40.20%. The cell-based assay showed a significant reduction in the cell viability, and NF-κB, TNF-α, and IL-6 levels upon treatment with CU-CHPNPs as compared to free CUR. Finally, the (CU-CHPNPs) downregulated the expression of the Bcl-2 anti-apoptotic gene more effectively than CUR and the CHPNPs comparing with the β Actin housekeeping gene. This study concluded that the nano-encapsulation of CUR significantly enhances its antitumor efficacy via inhibition of NF-κB, IL-6, and TNF-α and downregulation of Bcl-2.

Unfractionated heparin (UFH) is an anionic glycosaminoglycan that is widely used to prevent blood clotting. However, in certain cases, unwanted side effects can require it to be neutralized. Protamine sulfate (PS), a basic peptide rich in arginine, is the only approved antagonist for UFH neutralization. Many adverse reactions occur with the clinical application of PS, including systemic hypotension, pulmonary hypertension, and anaphylaxis. We previously described R15, a linear peptide composed of 15 arginine molecules, as a potential UFH antagonist. In this study, the in‐depth safety of R15 was explored to reveal its merits and associated risks in comparison with PS. In vitro safety studies investigated the interactions of R15 with erythrocytes, fibrin, complement, and rat plasma. In vivo safety studies explored potential toxicity and immunogenicity of R15 and the UFH–R15 complex. Results showed that both PS and R15 can induce erythrocyte aggregation, thicken fibrin fibers, activate complement, and cause anticoagulation in a concentration‐dependent manner. However, those influences weakened in whole blood or in live animals and were avoided when R15 was in a complex with UFH. We found dramatically enhanced complement activation when there was excess UFH in analyses involving UFH–PS complexes, and a slight increase in those involving UFH–R15 complexes. Within 2 h, R15 was degraded in rat plasma in vitro, whereas PS was not. Enhanced creatinine was found after a single intravenous injection of PS or R15 (900 U·kg−1, body weight), suggesting possible abnormal renal function. The UFH–PS complex, but not the UFH–R15 complex, exhibited obvious immunogenicity. In conclusion, R15 is nonimmunogenic and potentially safe at a therapeutic dose to reverse the effects of UFH., Safety studies of R15, a new heparin antagonist, were investigated in comparison with PS (protamine sulfate). R15 was degradable in plasma of rats within 2 h, whereas PS was not. Dramatically enhanced complement activation was observed for UFH–PS complex with excess UFH (unfractionated heparin). UFH–PS complex possessed immunogenicity that was completely absent for the UFH–R15 complex.

The selective cytotoxicity of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) to cancer cells but not to normal cells makes it an attractive candidate for cancer therapeutics. However, the disadvantages of TRAIL such as physicochemical instability and short half-life limit its further clinical applications. In this study, TRAIL was encapsulated into a novel anti-angiogenic nanocomplex for both improved drug distribution at the tumor site and enhanced anti-tumor efficacy. A nanocomplex was prepared firstly by entrapping TRAIL into PEG-low molecular weight heparin-taurocholate conjugate (LHT7), which is previously known as a potent angiogenesis inhibitor. Then, protamine was added to make a stable form of nanocomplex (PEG-LHT7/TRAIL/Protamine) by exerting electrostatic interactions. We found that entrapping TRAIL into the nanocomplex significantly improved both pharmacokinetic properties and tumor accumulation rate without affecting the tumor selective cytotoxicity of TRAIL. Furthermore, the anti-tumor efficacy of nanocomplex was highly augmented (73.77±4.86%) compared to treating with only TRAIL (18.49 ± 19.75%), PEG-LHT7/Protamine (47.84 ± 14.20%) and co-injection of TRAIL and PEG-LHT7/Protamine (56.26 ± 9.98%). Histological analysis revealed that treatment with the nanocomplex showed both anti-angiogenic efficacy and homogenously induced cancer cell apoptosis, which suggests that accumulated TRAIL and LHT7 in tumor tissue exerted their anti-tumor effects synergistically. Based on this study, we suggest that PEG-LHT7/Protamine complex is an effective nanocarrier of TRAIL for enhancing drug distribution as well as improving anti-tumor efficacy by exploiting the synergistic mechanism of anti-angiogenesis.

Packaging paternal genome into tiny sperm nuclei during spermatogenesis requires 10sup6/sup-fold compaction of DNA, corresponding to a 10-20 times higher compaction than in somatic cells. While such a high level of compaction involves protamine, a small arginine-rich basic protein, the precise mechanism at play is still unclear. Effective pair potential calculations and large-scale molecular dynamics simulations using a simple idealized model incorporating solely electrostatic and steric interactions clearly demonstrate a reversible control on DNA condensates formation by varying the protamine-to-DNA ratio. Microscopic states and condensate structures occurring in semidilute solutions of short DNA fragments are in good agreement with experimental phase diagram and cryoTEM observations. The reversible microscopic mechanisms induced by protamination modulation should provide valuable information to improve a mechanistic understanding of early and intermediate stages of spermatogenesis where an interplay between condensation and liquid-liquid phase separation triggered by protamine expression and post-translational regulation might occur. Moreover, recent vaccines to prevent virus infections and cancers using protamine as a packaging and depackaging agent might be fine-tuned for improved efficiency using a protamination control.

Background Epigenetics refers to an alteration in gene expression without alteration in the sequence of DNA and this process may be affected by environmental factors and lifestyle like cigarette smoking. This study was designed to evaluate the potential effect of cigarette smoking on the global DNA methylation status and the transcription level of protamine 1 and protamine 2 in human spermatozoa. A total of 188 semen samples were collected from men with a mean age of 34.9 ± 5.8 years old (98 heavy smokers and 90 non-smokers). The DNA and RNA were isolated from purified spermatozoa, then the status of global DNA methylation and the transcription level of protamine 1 and protamine 2 were evaluated using ELISA and qPCR, respectively. The chromatin non-condensation and DNA fragmentation in human spermatozoa were evaluated using chromomycin A3 staining and TUNEL assay, respectively. Results A significant increase has been found in the status of global DNA methylation in spermatozoa of heavy smokers compared to non-smokers (7.69 ± 0.69 ng/μl vs. 4.90 ± 0.40 ng/μl, P < 0.001). Additionally, a significant reduction has been found in transcription level of protamine 1 (25.49 ± 0.31 vs. 23.94 ± 0.40, P < 0.001) and protamine 2 (28.27 ± 0.39 vs. 23.45 ± 0.30, P < 0.001) in heavy smokers. A downregulation has been found in the transcription level of protamine 1 and protamine 2 with a fold change of 0.497 and 0.047, respectively. A significant increase has been shown in the level of DNA fragmentation and chromatin non-condensation in heavy smokers compared to non-smokers (P < 0.001). On the other hand, a significant positive correlation has been found between sperm chromatin non-condensation, sperm DNA fragmentation, transcription level of protamine 1, transcription level of protamine 2, and global DNA methylation status (r = 0.304, P < 0.001; r = 0.399, P < 0.001; r = 0.216, P = 0.003; r = 0.494, P < 0.001, respectively). Conclusion Tobacco cigarette smoking has a potential influence on the global DNA methylation and the transcription level of protamine genes in human spermatozoa, and consequently, affect negatively on the semen parameters.

DNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes (SMC) proteins such as condensin. Here, however, we are interested in a different looping method whereby multivalent cations (charge≥+3), such as protamine proteins, neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (≤ 1 μm) DNA fragments using atomic force microscopy (AFM). We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it overestimated the number of loops that form at the ends of the molecule and failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine’s in vivo role for looping DNA into toroids and the mechanism of DNA condensation by multivalent cations more broadly.SIGNIFICANCEDNA looping is important in a variety of both in vivo functions (e.g. gene regulation) and in vitro applications (e.g. DNA origami). Here, we sought a mechanistic understanding of DNA looping by multivalent cations (≥+3), which condense DNA into loops and toroids. One such multivalent cation is the protein protamine, which condenses DNA in sperm. We investigated the mechanism for loop formation by protamine and found that the experimental data was consistent with an electrostatic multibinding model in which two protamines bind electrostatically to the DNA within a 50-nm region to form a loop. This model is likely general to all multivalent cations and may be helpful in applications involving toroid formation or DNA nanoengineering.

The acetic acid-urea polyacrylamide gel electrophoresis system could separate very similar basic proteins on differences in size and effective charge. This system has been used for many years to analyse histones and their post-translational modifications and widely used in the study of mammal protamines. Two types of protamine have been described, the protamine 1 (P1) and the protamine 2 (P2) family members, which are synthetized by PRM1 and PRM2 genes. The ratio of P1 and P2 is important for predicting fertility in humans and mice. Therefore, the quantification of protamines is a fundamental step in order to establish the ratio between P1 and P2 in these species. In other mammals, studies linking sperm protamination and the protamine ratio with fertility are increasing. So, the use of an effective technique to separate and quantify protamines is important to study sperm P1/P2 ratio. Therefore, this article describes in detail a feasible and useful procedure to isolate bovine sperm protamines, to perform pre-electrophoresis with PEG solution and finally to carry out acid-urea polyacrylamide gel electrophoresis in reverse polarity. This technique allows a clear separation and efficient detection of bovine sperm protamines.

Studying thermal stability of proteins not only provides insight into protein structure but also is instrumental in identifying previously unknown interaction partners. We develop a machine learning strategy that combines orthogonal partial least squares regression and stability screening of Silver Bullets Bio library to identify biologically active molecules that enhance protein stability. This strategy proves effective in extracting the stability-enhancing molecules for SMYD5, a histone lysine methyltransferase that regulates chromosome integrity. Protamine, a histone substitute in chromatin condensation during spermatogenesis, is identified as the most influential molecule to enhance SMYD5 thermal stability. We find that the C-terminal poly-glutamic acid tract (poly-E) and a 30-residue insertion in MYND domain (M-insertion), which are unique to SMYD5, regulate the structural stability. However, protamine plays a dominant role in SMYD5 stability, and in the presence of protamine, the poly-E tract or M-insertion loses its ability to affect the stability. The stability-enhancing effect of protamine is SMYD5 specific, and for SMYD2, a closely related homolog, protamine exhibits opposite, destabilizing effects. We find that both SMYD5 and SMYD2 interact with protamine, where SMYD5 interaction is independent of the poly-E tract and M-insertion. Protamine not only helps provide insight into the structure-stability relationships of SMYD5, but also suggests a potential functional link of SMYD5 to spermatogenesis. SMYD5 is a ubiquitously expressed gene with the highest expression in testis, especially in the seminiferous ducts that contain germ cells. Thus, our study opens up avenues that could help delineate major mechanisms underlying chromatin dynamics during spermatogenesis.

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter‐pair‐based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate‐limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage‐dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1–2 events per second and long dwell times, in agreement with the liposome study. Applied‐field and steered molecular dynamics simulations added an atomistic view of the permeation events. It can be concluded that a concentration gradient of 1 μm Ptm leads to a translocation rate of about one molecule per second and per channel., Surprisingly, large peptides (Protamine, 5.1 kDa) can permeate through bacterial outer membrane channels. The use of fluorescence, electrophysiology, and all‐atom modeling allows to quantify the flux. This approach can be transferred to related problems.

Context Protamine is used ubiquitously in all cardiac surgeries for reversal of heparin. Risk of postoperative bleeding is increased with inadequate heparin reversal or due to anticoagulant side effects of protamine; hence, it is important to dose protamine properly. This study compares 80% protamine dose with full dose on postoperative bleeding and transfusion needs in OPCAB. Aims The aim of our study was to find whether lower dose of protamine could reduce postoperative bleeding and need for blood product transfusions in off pump coronary artery bypass grafting as compared to the regular dose of protamine. Settings and design This was a double-blinded randomised controlled trial where patients posted for off pump CABG meeting the inclusion criteria were included in the study. Methods and material Ninety patients were randomised to two groups, group F receiving full dose of protamine of 1 mg per mg heparin used, and group L received 0.8 mg per mg. Postoperative activated clotting time, bleeding at 1 h, 4 h, 24 h and total drainage till drains removal and blood product transfusion requirements were noted. Statistical analysis used: SPSS software. Results Both groups were matched in demographics, preoperative cessation of heparin and aspirin and platelet counts. Both groups received equal heparin dose, activated clotting time before protamine, activated clotting time post protamine in OT and ICU were equal as were the conduits used. There was no significant difference between the groups in post-operative drainage over time or in the need for blood product transfusions. Conclusions Eighty per cent of the dose of protamine can adequately reverse the heparin used during off pump cardiac surgery without any increase in incidence of postoperative bleeding or need for blood product transfusions.

Cardiopulmonary bypass (CPB) is associated with platelet dysfunction (PD), an important cause of postoperative bleeding. The etiology of PD is not completely understood. We mapped the platelets' function during CPB to determine the etiology of PD. Platelets activation, measured by procaspase activating compound-1 and P-selectin expression (CD62P), after activation by adenosine diphosphate and thrombin receptor activator peptide, were decreased by protamine. Changes during CPB were insignificant. Platelet-leukocyte aggregation was increased by CPB but not by protamine. Platelet apoptosis marker, annexin V, was increased by protamine. Changes during CPB were insignificant. Our findings demonstrate that protamine given after CPB plays a central role in PD and count decrease.

A new fluorene-pyridine oligoelectrolyte (OFP) is rationally proposed and readily synthesized via a simple one-pot Sonogashira approach. Hence, an unexpectedly small cationic oligomer nanosensor (i.e. OFPNPs, ∼ 1.2 nm in diameter) was conveniently fabricated owing to the enhanced flexibility endowed by the meta-substituted pyridyl unit. Inspiringly, this facile nanoplatform with low cytotoxicity favors the ultrasensitive fluorescence assay for heparin and protamine with a detection limit (LOD, S/N = 3) as low as 1.2 ng mL-1 and 0.5 ng mL-1, respectively, involving heparin-induced aggregation of OFPNPs through electrostatic interaction or competitive rebinding of protamine to heparin.

Protamine, a polycation, is biologically and medically relevant protein. Protamine exhibits a wide array of functions in biological processes like gene transfer, tissue and organogenesis, cell reproduction, etc. Medically, Protamine is the only clinically approved antidote for Heparin and is routinely used in various surgical interventions, and hence controlling Protamine dosing in patients is very crucial. Taking into account the medical significance of Protamine, designing simple, reliable and sensitive fluorescence sensors is highly desirable. In this work, we propose one such sensitive and reliable fluorescent sensor which is based on a template of dye-polyelectrolyte assembly constituting a molecular rotor dye, Thioflavin-T and an anionic synthetic polyelectrolyte, polystyrene sulfonate. The addition of Protamine, prompts drastic modulations in spectral features of dye-polyelectrolyte assembly which enables sensitive detection of Protamine in aqueous solution. Apart from sensitive detection, our sensing platform aids in highly selective sensing of Protamine compared to other proteins. Moreover, our sensor system is constructed on label-free, inexpensive, commercially available molecules posing as an advantage over other sensor systems which involve laborious synthesis protocols. Most importantly, our sensor template is able to sense Protamine in diluted serum sample, indicating the potential practical utility of our sensor system.

Aims The current study aimed to explore the expression of transient receptor potential A1 ion channels (TRPA1) in the rat ureter and to assess if TRPA1-active compounds modulate ureter function. Methods The expression of TRPA1 in rat ureter tissue was studied by immunofluorescence. The TRPA1 distribution was compared to calcitonin gene-related peptide (CGRP), alpha-actin (SMA1), anoctamin-1 (ANO1), and c-kit. For in vivo analyses, a catheter was implanted in the right ureter of 50 rats. Ureter peristalsis and pressures were continuously recorded by a data acquisition set-up during intraluminal infusion of saline (baseline), saline plus protamine sulfate (PS; to disrupt the urothelium), saline plus PS with hydrogen sulfide (NaHS) or cinnamaldehyde (CA). Comparisons were made between rats treated systemically with vehicle or a TRPA1-antagonist (HC030031). Results TRPA1-immunoreactive nerves co-expressed CGRP and were mainly located in the suburothelial region of the ureter. Immunoreactivity for TRPA1 was also encountered in c-kit-positive but ANO1-negative cells of the ureter suburothelium and wall. In vivo, HC030031-treated rats had elevated baseline peristaltic frequency (p < 0.05) and higher intraluminal pressures (p < 0.01). PS increased the frequency of ureter peristalsis versus baseline in vehicle-treated rats (p < 0.001) but not in HC030031-treated rats. CA (p < 0.001) and NaHS (p < 0.001) decreased ureter peristalsis. This was counteracted by HC030031 (p < 0.05 and p < 0.01). Conclusions In rats, TRPA1 is expressed on cellular structures considered of importance for peristaltic and mechanoafferent functions of the ureter. Functional data indicate that TRPA1-mediated signals regulate ureter peristalsis. This effect was pronounced after mucosal disruption and suggests a role for TRPA1 in ureter pathologies involving urothelial damage. Funding Agencies|Gester Foundation, Helsingborg, Sweden

Protamine causes cardiac depression, which may be mediated by tumor necrosis factor alpha (TNF-α). Ulinastatin, a human urinary protease inhibitor, inhibits TNF-α. Here, we aimed to investigate whether ulinastatin prevented protamine-induced myocardial depression by inhibiting TNF-α. Rat hearts were perfused using a Langendorff system, and three protocols were followed. Protocol 1: The hearts were divided into saline, ulinastatin-low, and ulinastatin-high groups. Protamine was administered to each group, and myocardial contractility was the primary outcome. Protocol 2: The hearts were allotted to saline or ulinastatin group. Protamine was administered to each group. TNF-α expression in the coronary effluent and myocardial tissue was measured. Protocol 3: The hearts were allotted to saline and ulinastatin groups. Recombinant rat-TNF-α was administered to each group. Protamine alone reduced the maximum left ventricular pressure derivative (LV dP/dt max) by 45 ± 4%. In contrast, the reduction in LV dP/dt max was 4 ± 3% in the ulinastatin-high group. Compared with that in the saline group, the increase in TNF-α in the coronary effluent was attenuated in the ulinastatin group. Recombinant TNF-α alone reduced LV dP/dt max (- 21 ± 14%). In contrast, when TNF-α was added in the presence of ulinastatin, the decrease in LV dP/dt max was prevented significantly (- 3 ± 8%). We showed, for the first time, that ulinastatin protected against protamine-induced myocardial damage, both by inhibiting TNF-α synthesis and by directly preventing the cardiodepressant action of TNF-α.

Background: The aim of this study was to evaluate risk factors associated with difficult heparin reversal by protamine after cardiopulmonary bypass.Methods: Data from 120 consecutive patients who underwent open heart surgery from 2009 to 2017 were retrospectively reviewed. Patients were divided into 2 groups: (1) those in whom complete heparin reversal was achieved after a single infusion of protamine (group A, n=89); and (2) those who required more protamine for heparin reversal (group B, n=31).Results: Female sex, prolonged bypass time (>200 min), long aortic cross-clamping time (>120 min), and a lowest rectal temperature

Diabetes Mellitus (DM) is the most common endocrine disorder affecting many human physiological systems and tissues, including the reproductive organs in men. The age of individuals suffering from this disease has been falling rapidly in recent years. This study compared the effect of DM on sperm parameters, chromatin quality, and apoptosis, as well as the expression profile of protamine genes in men with and without DM using molecular and cytochemical assays. Sixty semen samples from the control group (

Glycerol is used as a bovine semen osmotic cryoprotectant that greatly improves the quality of frozen and thawed bovine sperm. However, high glycerol concentrations can have a toxic effect on frozen and thawed bovine sperm. Therefore, this experiment investigated the effect of replacing a portion of the glycerol in a cryoprotectant solution with crocin on the sperm apoptosis, protamine deficiency and membrane lipid oxidation of frozen and thawed Yanbian yellow cattle sperm. The experiment included a control group (6% glycerol) and four treatment groups: I (3% glycerol), II (3% glycerol +0.5 mM crocin), III (3% glycerol + 1 mM crocin) and IV (3% glycerol + 2 mM crocin). Computer assisted semen analysis was used to detect sperm motility, Hoechst 33,342, propidium iodide, and JC-1 staining were used to analyse sperm viability and mitochondrial membrane potential, chromomycin A3 staining was used to detect protamine deficiency and DNA damage, flow cytometry was used for sperm membrane lipid disorder detection and analysis, and real-time quantitative RT-qPCR was used to detect the mRNA expression levels of protamine-related genes (PRM2, PRM3), sperm acrosome-associated genes (SPACA3), oxidative stress-related genes (ROMO1) and apoptosis-related genes (BCL2, BAX). Compared to the control group, replacing a portion of glycerol with 1 mM crocin significantly improved sperm motility, plasma membrane integrity, membrane lipid disorders (p < .05) and viability, mitochondrial membrane potential, protamine deficiency (p < .01). The expression level of PRM2, PRM3, SPACA3 and BCL2 significantly increased (p < .05), while the expression levels of ROMO1 and BAX significantly decreased (p < .05). Accordingly, the BCL2/BAX ratio significantly increased (p < .05). In summary, the substitution of a portion of glycerol with crocin in cryoprotective solution improved the quality of Yanbian yellow cattle sperm after freezing and thawing.

OBJECTIVES In cardiac surgery, adequate heparinization is necessary to prevent thrombus formation in the cardiopulmonary bypass (CPB). To counteract the heparin effect after weaning from CPB, protamine is administered. The optimal protamine/heparin ratio is still unknown. METHODS In this before–after study, we evaluated the effect of a 0.6/1-protamine/heparin ratio implementation as of May 2017 versus a 0.8/1-protamine/heparin ratio on the 12-h postoperative blood loss and the amount of blood and blood component transfusions (fresh frozen plasma, packed red blood cells, fibrinogen concentrate, platelet concentrate and prothrombin complex concentrate) after cardiac surgery. A total of 2051 patients who underwent cardiac surgery requiring CPB between May 2016 and May 2018 were included. RESULTS In the 0.6/1-protamine/heparin ratio group, only 28.8% of the patients received blood component transfusion, compared to 37.9% of the patients in the 0.8/1-ratio group (P < 0.001). The median 12-h postoperative blood loss was 230 ml (interquartile range 140–320) in the 0.6/1-ratio group versus 260 ml (interquartile range 155–365) in the 0.8/1-ratio group (P < 0.001). CONCLUSIONS A 0.6/1-protamine/heparin ratio after weaning from CPB is associated with a significantly reduced 12-h postoperative blood loss and blood components transfusion.

Objectives While the use of protamine sulfate as a heparin reversal agent has been extensively reviewed in patients undergoing carotid endarterectomy and coronary artery bypass grafting, there is a lack of literature on protamine’s effects on lower extremity bypasses. The purpose of this study was to determine the risk of protamine sulfate dosing after tibial bypass on thrombotic or bleeding events, including early bypass failure. Methods We performed a retrospective review of our institutional database for patients undergoing primary distal peripheral bypass from January 2009 through December 2015 (contralateral bypass was considered to be a new primary bypass). Primary endpoints include composite thrombotic events (myocardial infarction, stroke, amputation at 30 days and patency less than 30 days) and composite bleeding events (bleeding or transfusion). Results A total of 152 tibial or peroneal bypasses in 136 patients with critical limb ischemia were identified. Of these, 78 (57.4%) patients received protamine sulfate intraoperatively and 58 (42.6%) did not. There were no differences in composite thrombotic or hemorrhagic outcomes. Protamine use had no effect on the rates of perioperative MI (9.0% versus 3.5%, p = 0.20), stroke (1.3% versus 1.7%, p = 0.83), or perioperative mortality (5.1% versus 3.5%, p = 0.64). There was no significant difference in composite post-operative bleeding events (20.7% versus 14.1%, p = 0.31) or composite thrombotic events (17.2% versus 18.0%, p = 0.91). Patients who received protamine undergoing bypass with non-autogenous conduit had significantly higher-recorded median operative blood loss (250 mL versus 150 mL, p = 0.0097) and median procedure lengths (265 min versus 201 min, p = 0.0229). No difference in 30-day amputation-free survival was noted (91.0% versus 91.4%, p = 0.94). Follow-up Kaplan–Meier estimation did not demonstrate a difference in 30-day patency (91.7% versus 88.5%, p = 0.52). Conclusions Heparin reversal with protamine sulfate after tibial or peroneal bypass grafting is not associated with higher cardiovascular morbidity, bypass thrombosis, amputation, or mortality. Additionally, there was no statistically significant difference in post-operative bleeding or thrombosis complications for patients who did not receive protamine, although the findings are suggestive of a potential difference in a more adequately powered study. Our results suggest that protamine sulfate is safe for intraoperative use without increased risk of thrombotic complications or early tibial bypass graft failure.

Objective: Since sperm abnormalities are known to be a major reason for recurrent pregnancy loss (RPL), any defects in DNA structure and chromatin condensation can place embryos at risk in the early stage of development and implantation. As antioxidants such as vitamin C may play a protective role against the destruction of protamine genes in sperm chromatin, this study was conducted to evaluate the effects of vitamin C on chromatin and the expression of protamine genes in the male partners of couples with RPL.Methods: Twenty male partners of couples with RPL were selected as the intervention group and received vitamin C supplementation (250 mg daily for 3 months). Healthy fertile men (n=20) were included as controls. Sperm chromatin, DNA integrity, and the expression levels of protamine genes were evaluated before and after treatment.Results: Significant differences were found in sperm morphology, protamine deficiency, and apoptosis between the two groups and before and after vitamin C administration. A significant change was found in mRNA levels of PRM1, PRM2, and the PRM1/PRM2 ratio after treatment.Conclusion: Daily oral administration of vitamin C may improve human sperm parameters and DNA integrity by increasing protamine gene expression levels in the male partners of couples with RPL. The beneficial effects of vitamin C supplementation as an antioxidant for the male partners of couples with RPL could lead to improved pregnancy outcomes in these cases.

Assisted reproductive technologies involving the use of spermatozoa and eggs for in vitro fertilization (IVF) have come as the solution for many infertile couples to become parents. However, in some cases, the use of ejaculated spermatozoa delivers poor IVF performance. Some studies have suggested the use of testicular spermatozoa in severe male infertility cases, but no guidelines regarding their utilization are currently available. In the present study, we found the mRNA protamine 1/protamine 2 (P1/P2) ratio to be a valuable biomarker of poor sperm function that could be used as a diagnostic key for the identification of cases that would benefit from the use of testicular spermatozoa. A total of 23 couples undergoing egg donation cycles with at least one previous cycle failure were studied. All couples underwent two consecutive intracytoplasmic sperm injection (ICSI) cycles with either ejaculated or testicular spermatozoa (TESA). The sperm mRNA P1/P2 ratio, fertilization rate, blastocyst rate, and pregnancy and live birth rate were compared. Results showed improved ICSI and clinical outcomes in cycles with testicular spermatozoa in men with altered mRNA P1/P2 ratios. TESA cycles presented significantly higher rates of fertilization (mean ± standard deviation: 76.1% ± 15.1% vs 65.5% ± 18.8%), blastocyst formation (55.0% ± 20.3% vs 30.8% ± 23.8%), and good morphological quality blastocyst (28.9% ± 22.9% vs 13.5% ± 17.9%) and also improvements on pregnancy (60.9% vs 0%) and healthy birth rates (56.5% vs 0%) than EJACULATE cycles. The results described here suggest that in patients with previous IVF/ICSI failures and aberrant mRNA protamine ratios, the use of testicular spermatozoa may be a good alternative to improve clinical outcomes.

OBJECTIVE To assess effects of basal-bolus insulin treatment (BBIT) with lispro and neutral protamine Hagedorn (NPH) insulins, compared with NPH insulin alone, on serum fructosamine concentration (SFC) and postprandial blood glucose concentration (BGC) in dogs with clinically well-controlled diabetes mellitus and postprandial hyperglycemia fed a high insoluble fiber–content diet. ANIMALS 6 client-owned dogs with diabetes mellitus. PROCEDURES Blood samples were collected for BGC and SFC measurement in hospitalized dogs just before feeding and routine SC NPH insulin administration (time 0); samples were collected for BGC measurement every 30 minutes for 2 hours, then every 2 hours for up to 10 additional hours. Postprandial hyperglycemia was identified when BGC 30 minutes after insulin administration exceeded BGC at time 0 or the 1-hour time point. For BBIT, owners were instructed to continue NPH insulin administration at the usual dosage at home (q 12 h, with feeding) and to administer lispro insulin (0.1 U/Kg, SC) separately at the time of NPH injections. Two weeks later, SFC and BGC measurements were repeated; results at the start and end of the study were compared statistically. RESULTS Median SFC was significantly higher at the start (400 μmol/L) than at the end (390 μmol/L) of the study. Median 1-hour (313 mg/dL) and 1.5-hour (239 mg/dL) BGC measurements at the start of the study were significantly higher than those at the end of the study (117 and 94 mg/dL, respectively). CONCLUSIONS AND CLINICAL RELEVANCE In this sample of dogs with well-controlled diabetes mellitus, addition of lispro insulin to an existing treatment regimen of NPH insulin and dietary management significantly decreased postprandial BGCs. Further study of BBIT for dogs with diabetes mellitus is warranted.

A label-free, sensitive, simple and general colorimetric method was reported to monitor S1 nuclease activity based on protamine-assisted aggregation of gold nanoparticles (AuNPs). Here, protamine, a linear polycation, was used as a medium for causing the aggregation of negatively charged AuNPs by electrostatic interactions, resulting in changes in the surface plasmon resonance (SPR) absorption bands as well as the color of AuNPs. Here, the AuNPs were employed as an indicator to detect the level of S1 nuclease in the solution. Substrate DNA could be cleaved into small fragments by the specific S1 nuclease, which effectively prevents the electrostatic interaction between DNA and protamine and thus facilitates the interaction between protamine and AuNPs. The quantitative analysis of S1 nuclease activity can be performed via directly measuring the changes in the absorption spectra of the AuNPs. Using S1 nuclease as a model analyte, the limit of detection was estimated to be 1.0 × 10-4 U mL-1. Furthermore, the proposed concept has been successfully applied in S1 nuclease analysis of serum samples, offering an ultrasensitive strategy for the speedy detection of the nuclease activity and providing a new avenue for high-throughput screening of nucleases and drugs with potential inhibition properties.

Objectives The aim of this study was to evaluate polymorphisms of sperm protamine genes and their effects on the result of CMA3 staining in varicocele men. Material and methods In a case control study, 128 patients with male infertility due to varicocele and 128 controls were recruited. Polymorphisms of PRM1 and PRM2 genes in extracted DNA samples were assessed by PCR-SSCP and sequencing. Protamine deficiency was also indirectly estimated by CMA3 staining. Result Nine different variants including six variants in PRM1 gene and three variants in PRM2 gene were found among varicocele patients. The results showed that sperm count, motility and morphology were significantly different between control group without gene variations and varicocele group who had several variations in their protamine genes (P Conclusion Therefore, PRM1 and PRM2 variations in varicocele patients are associated with the production of spermatozoa with more protamine deficiency and this is one of the possible causes of infertility due to varicocele.

The management of a patient with hemophilia undergoing cardiovascular surgery relies on accurate coagulation test results. Both unfractionated heparin (UFH) and protamine sulfate used during cardiac surgery can interfere with factor and inhibitor assays. Here we describe the effects of UFH and protamine sulfate on routine coagulation, factor activity, and inhibitor assays. Pooled normal plasma (PNP) with UFH, PNP with protamine sulfate, PNP with both protamine sulfate and UFH were tested for the activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), UFH anti-Xa, one-stage factor VIII (FVIII) activity, one-stage factor IX (FIX) activity, and Bethesda inhibitor assays for FVIII and FIX. UFH had a dose-dependent effect with TT, aPTT, and PT. On Bethesda inhibitor testing, FIX inhibition was detected at 1 U/ml UFH and 3 U/ml UFH for FVIII. Increasing protamine sulfate concentration in PNP prolonged the PT and aPTT in a dose-dependent manner, decreased FVIII and FIX activity and did not affect TT or UFH anti-Xa. At protamine sulfate doses of at least 200 μg/ml there was weak FVIII and FIX inhibition detected. At lower ratios of protamine sulfate to UFH (0.6 : 1-0.8 : 1), the aPTT decreased, suggesting reversal of UFH. However, at protamine sulfate to UFH ratios of 1.0 : 1 and higher, aPTT prolongation was observed. Inhibition of FVIII and FIX was detected at low ratios of protamine sulfate to UFH (below 0.4 : 1) and disappeared at higher ratios. UFH and protamine sulfate, alone or in combination, impact factor activity and inhibitor testing for both FVIII and FIX. Hence, factor activity and inhibitor assay results should be interpreted with caution when UFH or protamine sulfate are present.

Uncontrolled bleeding after enoxaparin (ENX) is rare but may be life-threatening. The only registered antidote for ENX, protamine sulfate (PS), has 60% efficacy and can cause severe adverse side effects. We developed a diblock copolymer, heparin-binding copolymer (HBC), that reverses intravenously administered heparins. Here, we focused on the HBC inhibitory activity against subcutaneously administered ENX in healthy mice. BALB/c mice were subcutaneously injected with ENX at the dose of 5 mg/kg. After 110 min, vehicle, HBC (6.25 and 12.5 mg/kg), or PS (5 and 10 mg/kg) were administered into the tail vein. The blood was collected after 3, 10, 60, 120, 360, and 600 min after vehicle, HBC, or PS administration. The activities of antifactors Xa and IIa and biochemical parameters were measured. The main organs were collected for histological analysis. HBC at the lower dose reversed the effect of ENX on antifactor Xa activity for 10 min after antidote administration, whereas at the higher dose, HBC reversed the effect on antifactor Xa activity throughout the course of the experiment. Both doses of HBC completely reversed the effect of ENX on antifactor IIa activity. PS did not reverse antifactor Xa activity and partially reversed antifactor IIa activity. HBC modulated biochemical parameters. Histopathological analysis showed changes in the liver, lungs, and spleen of mice treated with HBC and in the lungs and heart of mice treated with PS. HBC administered in an appropriate dose might be an efficient substitute for PS to reverse significantly increased anticoagulant activity that may be connected with major bleeding in patients receiving ENX subcutaneously.

Perioperative myocardial infarction is a serious complication affecting a significant portion of patients undergoing coronary artery bypass graft surgery. This may arise due to coronary graft thrombosis, a rare but potentially fatal phenomenon associated with both congenital and acquired risk factors. Multiple case reports implicate the role of protamine in the development of such thromboses. The role of protamine in facilitating the regulation of hemostasis by reversing the anticoagulant effects of heparin in patients undergoing cardiopulmonary bypass is well-recognized. However, discussion of its potential contribution to coronary graft thrombosis and mechanisms by which this may occur is lacking. Furthermore, its narrow therapeutic index and side effect profile are such that its appropriateness as a universal reversal agent to heparin requires reconsideration. This article reviews the current body of evidence regarding the use of protamine in cardiac surgery and the limited case reports pertaining to its potential role in the pathophysiology of coronary graft thrombosis.

Background Intraoperative graft assessment with tools like Transit Time Flow Measurement (TTFM) is imperative for quality control in coronary surgery. We investigated the variation of TTFM parameters before and after protamine administration to identify new benchmark parameters for graft quality assessment. Methods The database of the REQUEST (“REgistry for QUality AssESsmenT with Ultrasound Imaging and TTFM in Cardiac Bypass Surgery”) study was retrospectively reviewed. A per graft analysis was performed. Only single grafts (i.e., no sequential nor composite grafts) where both pre- and post-protamine TTFM values were recorded with an acoustical coupling index > 30% were included. Grafts with incomplete data and mixed grafts (arterio-venous) were excluded. A second analysis was performed including single grafts only in the same MAP range pre- and post- protamine administration. Results After adjusting for MAP, we found a small increase in MGF (29 mL/min to 30 mL/min, p = 0.009) and decrease in PI (2.3 to 2.2, p Conclusion The small changes in TTFM parameters observed before and after protamine administration seem to be clinically irrelevant, despite being statistically significant in aggregate. Our data do not support a need to perform TTFM measurements both before and after protamine administration. A single TTFM measurement taken either before or after protamine may suffice to achieve reliable data on each graft’s performance. Depending on the specific clinical situation and intraoperative changes, more measurements may be informative. Trial registration Clinical Trials Number: NCT02385344, registered February 17th, 2015.

Dietary protamine can ameliorate hyperlipidemia; however, the protamine-derived active peptide and its hypolipidemic mechanism of action are unclear. Here, we report the discovery of a novel anti-obesity and hypocholesterolemic peptide, RPR (Arg-Pro-Arg), derived from protamine in mice fed a high-fat diet for 50 days. Serum cholesterol levels were significantly lower in the protamine and RPR groups than in the control group. White adipose tissue weight was significantly decreased in the protamine and RPR groups. The fecal excretion of cholesterol and bile acid was significantly higher in the protamine and RPR groups than in the control group. We also observed a significant decrease in the expression of hepatic SCD1, SREBP1, and adipocyte FAS mRNA, and significantly increased expression of hepatic PPARα and adipocyte PPARγ1 mRNA in the protamine group. These findings demonstrate that the anti-obesity effects of protamine are linked to the upregulation of adipocyte PPARγ1 and hepatic PPARα and the downregulation of hepatic SCD1 via SREBP1 and adipocyte FAS. RPR derived from protamine has a crucial role in the anti-obesity action of protamine by evaluating the effective dose of adipose tissue weight loss.

The association of PRM1/2 with male azoospermia is well-documented, but the relationship between TXNDC2 deficiency and the azoospermia phenotype, sperm retrieval, and pathology has not been elucidated. Here we identified the association of TXNDC2 and protamines in evaluating testis pathology and sperm retrieval. An extensive microarray meta-analysis of men with idiopathic azoospermia was performed, and after undergoing several steps of data quality controls, the data passing QC were pooled and batch effect corrected. As redox imbalance has been shown to have a variable relationship with fertility, our relative expression studies began with candidate protamination and thioredoxin genes. We constructed a logistic regression model of TXNDC2 with PRM1 and PRM2 genes, and collective ROC analysis indicated a sensitivity of 96.8% and specificity of 95.5% with a ROC value of 0.995 (SE = 0.0070, 95% CI 0.982–1.000). These results demonstrate that TXNDC2, PRM1, and PRM2 combined have a robust power to predict sperm retrieval and correlate with severe azoospermia pathology.

Protamines are small, highly-specialized, arginine-rich, and intrinsically-disordered chromosomal proteins that replace histones during spermiogenesis in many organisms. Previous evidence supports the notion that, in the animal kingdom, these proteins have evolved from a primitive replication-independent histone H1 involved in terminal cell differentiation. Nevertheless, a direct connection between the two families of chromatin proteins is missing. Here, we primarily used electron transfer dissociation MS-based analyses, revealing that the protamines in the sperm of the liverwort Marchantia polymorpha result from post-translational cleavage of three precursor H1 histones. Moreover, we show that the mature protamines are further post-translationally modified by di-aminopropanelation, and previous studies have reported that they condense spermatid chromatin through a process consisting of liquid-phase assembly likely involving spinodal decomposition. Taken together, our results reveal that the interesting evolutionary ancestry of protamines begins with histone H1 in both the animal and plant kingdoms.

Among the diverse sensing techniques, fluorimetric detection dominates over the other methods because of its rapid signaling, high selectivity and sensitivity, and operational simplicity. This present article delineates fabrication of a fluorescent organic nanoparticle-protamine (FONP-Pro) conjugate for selective and sensitive detection of heparin simply by exploitation of the aggregation-induced emission (AIE) property of the FONPs. Naphthalene diimide-based bola-type amphiphilic molecules (NDI

Background The monitoring of unfractionated heparin (UFH) reversal with protamine plays a crucial role for bleeding management after cardio-pulmonary bypass (CPB) in congenital cardiac surgery. The current standard for the monitoring of UFH and its reversal is the activated clotting time (ACT). While the ACT is affected by other CPB-associated pathologies a bedside technique with more specific heparin-related results would be very helpful. The new point-of-care viscoelastic test Haemonetics TEG® 6s, which is based on small blood samples may fulfill these requirements. This study aimed to compare the new TEG with laboratory assays. Methods A retrospective observational study was performed on 40 children with a median age of 130 days (interquartile range 13 to 310 days) undergoing congenital cardiac surgery. After separation of CPB, test results of the TEG® 6s, ACT, anti-Xa for UFH and PTT were compared and correlated with each other. Results No clinically relevant correlation was found for heparin specific TEG-derived parameters (CK/CKH R-time ratio) with ACT, PTT and anti-Xa measurements. After grouping in dependence to the CK/CKH R-time in patients with and without successful heparin reversal again no significant difference of anti-Xa-UFH-levels, post-/pre-CPB ratio of the PTT and ACT was observed. Conclusions In pediatric patients undergoing cardiac surgery using CPB there is no association of conventional coagulation tests and TEG-derived results. While bedside viscoelastic tests deliver rapid results, further studies are needed to compare whether the TEG based management of incomplete heparin reversal is sufficient to monitor heparin reversal and to reduce blood loss.

Background Bisphenol A is widely used in the manufacture of polycarbonate plastics and has caused increasing concern over its potential adverse impacts on spermatogenesis. However, the effect of bisphenol A on spermiogenesis is yet to be explored. Objectives To evaluate whether bisphenol A has adverse effects on DNA integrity and protamination of spermatogenic cell. Materials and methods Newborn male mice were subcutaneously injected with bisphenol A (0.1, 5 mg/kg body weight, n = 15) or coin oil (control group, n = 20) daily from post-natal day 1 until 35. At post-natal day 70, epididymis caudal spermatozoa and testes were collected. Sperm count, sperm motility, and sperm morphology were analyzed. The sperm chromatin structure assay was performed to examine the sperm DNA fragmentation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method was used to assess apoptosis of spermatogenic cells. The ultrastructural features of testicular sections were examined under a transmission electron microscope. Western blot and RT-PCR were used to detect the expression levels of transition protein (Tnp) 1 and Tnp2, protamine (Prm) 1 and Prm2 protein, and mRNA in mice testes. Results Bisphenol A significantly reduced sperm counts, impaired sperm motility, and increased the percentage of malformed spermatozoa. Poor sperm chromatin integrity and increased TUNEL-positive spermatogenic cells were also observed in mice exposed to bisphenol A. Ultrastructural analysis of testes showed that bisphenol A exposure caused incomplete chromatin condensation, retention of residual cytoplasm, and abnormal acrosome formation. In addition, the relative expression levels of Tnp2 and Prm2 in mice testes decreased significantly in bisphenol A groups. Discussion and conclusion Our findings identified that neonatal bisphenol A exposure may negatively contribute to the sperm quality in adult mice. Mechanistically, we showed that bisphenol A reduced sperm chromatin integrity along with increased DNA damage, which may be due to poor protamination of spermatozoa.

Objectives: Protamine reduces platelet aggregation after cardiopulmonary bypass (CPB). We studied the inhibitory effect of a reduced protamine dose, the duration of impaired platelet function and the possible correlation to postoperative bleeding. Design: Platelet function was assessed by impedance aggregometry in 30 patients undergoing cardiac surgery with CPB at baseline, before protamine administration, after 70% and 100% of the calculated protamine dose, after 20 minutes and at arrival to the intensive care unit. Adenosine diphosphate (ADP), thrombin receptor activating peptide-6 (TRAP), arachidonic acid (AA) and collagen (COL) were used as activators. Blood loss was measured during operation and three hours after surgery. Results are presented as median (25th-75th percentile). Results: Platelet aggregation decreased markedly after the initial dose of protamine (70%) with all activators; ADP 89 (71-110) to 54 (35-78), TRAP 143 (116-167) to 109 (77-136), both p amp;lt; .01; AA 25 (16-49) to 17 (12-24) and COL 92 (47-103) to 60 (38-81) U, both p amp;lt; .05. No further decrease was seen after 100% protamine. The effect was transient and after twenty minutes platelet aggregation had started to recover; ADP 76 (54-106), TRAP 138 (95-158), AA 20 (10-35), COL 70 (51-93) U. Blood loss during operation correlated to aggregometry measured at baseline and after protaminization. Conclusions: Protamine after CPB induces a marked decrease in platelet aggregation already at a protamine-heparin ratio of 0.7:1. The impairment seems to be transient and recovery had started after 20 minutes.

MicroRNAs (miRNAs) play a key role on gene expression regulation contributing to cell homeostasis, and they are highly dysregulated in cancer. Consequently, miRNA-based therapies are an attractive approach to develop novel anticancer strategies. The main objective of this work was to explore the full potential of protamine nanocapsules (Pr NCs) to develop an anticancer therapy based on the restoration of oncosuppressor miR-145, downregulated in colorectal cancer cells. The composition of Pr NCs was defined based on the selection of surfactants, and protamine that would enable an efficient association and intracellular delivery of miRNA mimics according to the layer-by-layer approach, and the encapsulation of curcumin within the oily core. After exposure of colorectal cancer cells with (i) miR-145 and (ii) curcumin-loaded Pr NCs, a strong increase in the intracellular levels of miR-145, which translated into a decreased cell proliferation rate and migration capacity of the treated cells, was observed. The potential of exploiting Pr NCs for the co-delivery of both biomolecules, miRNAs and curcumin, has also been proved. All together, here we evaluate the possibility to use Pr NCs to efficiently increase the intracellular levels of the oncosuppressor miR-145.

Objectives A hemostasis management system (HMS) is a point-of-care method for heparin and protamine titration. The authors hypothesized that protamine dosing over the HMS estimate would be associated with elevated activated clotting time (ACT), increased bleeding, and transfusion owing to protamine's anticoagulant activity. Design A retrospective cohort study. Setting Single-center university hospital. Participants One hundred eighty-nine patients undergoing elective coronary artery bypass grafting surgery. Interventions Patients were stratified into 3 groups per ratio of actual total administered protamine versus the HMS-derived protamine estimate: (1) low-ratio (≤66% of HMS estimate), (2) moderate-ratio (66%-100% of HMS estimate), and (3) high-ratio (>100% of HMS estimate). Measurements and Main Results The primary endpoints were post-protamine ACT, and residual heparin levels on HMS among the 3 groups in addition to bleeding and transfusion. There were 54 (28.6%) patients in the low, 95 (50.3%) in the moderate, and 40 (21.2%) in the high-ratio group. The high-ratio patients who were overdosed with protamine relative to the HMS estimate had elevated ACT, international normalized ratio, and activated partial thromboplastin time values, and subsequently received more red blood cell (RBC) and non-RBC transfusions compared to lower-ratio groups. Higher actual/HMS protamine ratios were associated independently with post-protamine ACT elevations after adjustment for sex, body mass index (BMI), and cardiopulmonary bypass (CPB) time. Conclusion Most patients received the protamine dose sufficiently close to the HMS estimate, but protamine dosing above the HMS estimate occurred in both obese and nonobese patients, which was associated independently with prolonged ACT after adjusting for sex, BMI, and CPB time.

Heparin and protamine are two indispensable agents of cardiopulmonary bypass surgery with effects on the cardiovascular and hematological system. Heparin is used as an anticoagulant in open heart surgery; whereas protamine is used to neutralize heparin effects when surgery is terminated. Protamine is given in order to neutralize heparin effects after cardiopulmonary bypass surgery and it causes hypotension in patients. However, the mechanism of this side effect is not clearly known. Current mechanism is that hypotension occurs after the administration of protamine due to the conformational change in the calcium channels or anaphylactoid thromboxane release or serum ionized calcium levels. The present study was to explain how protamine causes hypotension in evidence-based medicine indexed in PubMed and Web of Science. In addition to above mechanisms, possibly the infused protamine binds heparin and causes the coagulation cascade to activate heparin-AT complex on thrombin beside activating FXIa, FXa and FIXa and causing the re-use of Ca2+. The re-use of Ca2+ at the coagulation cascade initiates an anion gap and it is assumed that hypotension develops because of the Ca2+ deficiency. Ca2+ ions are trapped in the thrombus by the resumption of thrombus formation. Ca2+ ions trapped in the thrombus cannot be used, so that Ca2+ ion deficit will develop in circulation and hypotension occurs due to the insufficiency of Ca2+ ions. The administration of Ca2+ ions together with the protamine might help to eliminate the side effect of the protamine (hypotension) while neutralizing heparin after open heart surgery in light of the information provided in the literature.

Recently, many researchers have reported that DNA nanostructures, such as tetrahedral framework nucleic acids (tFNAs), have great potential to be useful tools in clinical and laboratory applications due to their programmable shapes, functional sites, and biological responses. However, finite endocytosis and stability in cells and body fluids compromise the functions of DNA nanostructures as a result of various adverse factors. In this study, we successfully synthesized PEGylated protamine, and tFNAs were adsorbed to it in a proper ratio of nitrogen in protamine to phosphorus in tFNAs (N/P ratio) as the functional complex. Furthermore, we demonstrated that PEGylated protamine-adsorbed tFNAs show a more prominent positive effect on cell viability and proliferation than naked tFNAs do. An increase in endocytosis can be observed in three different tissue-derived cells with the PEG-protamine-tFNA (PPT) complex. The increased endocytic ability is mediated by multiple pathways; moreover, the stimulatory effect of the PPT complex on the endocytic ability is dramatically blocked by the inhibition of the caveola-dependent pathway. Consistently, when tFNAs are stabilized by PEGylated protamine, they often tend to escape from lysosomes and survive for a longer period in biological fluids rather than being rapidly eliminated from the kidneys. The in vitro and in vivo results of our study demonstrate that the PPT complex method is a feasible, potent, and low-cost strategy that improves tFNA biocompatibility, stability, and internalization. This study provides evidence supporting the possibility of implementing PPTs for use in drug delivery, bioimaging, and gene transfection in the future.

Jun-Pil Jee1,*, Rudra Pangeni2,*, Saurav Kumar Jha,2 Youngro Byun,3 Jin Woo Park21College of Pharmacy, Chosun University, Gwangju 61452, Republic of Korea; 2College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Muan-gun, Jeonnam 58554, Republic of Korea; 3Department of Molecular Medicine and Biopharmaceutical Science, Graduate School of Convergence Science and Technology, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea*These authors contributed equally to this workPurpose: We created and evaluated an enhanced topical delivery system featuring a combination of highly skin-permeable growth factors (GFs), quercetin (QCN), and oxygen; these synergistically accelerated re-epithelialization and granulation tissue formation of/in diabetic wounds by increasing the levels of GFs and antioxidants, and the oxygen partial pressure, at the wound site.Methods: To enhance the therapeutic effects of exogenous administration of GFs for the treatment of diabetic wounds, we prepared highly skin-permeable GF complexes comprised of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), platelet-derived growth factor-A (PDGF-A), and basic fibroblast growth factor (bFGF), genetically attached, via the N-termini, to a low-molecular-weight protamine (LMWP) to form LMWP-EGF, LMWP-IGF-I, LMWP-PDGF-A, and LMWP-bFGF, respectively. Furthermore, quercetin (QCN)- and oxygen-carrying 1-bromoperfluorooctane (PFOB)-loaded nanoemulsions (QCN-NE and OXY-PFOB-NE) were developed to improve the topical delivery of QCN and oxygen, respectively. After confirming the enhanced penetration of LMWP-GFs, QCN-NE, and oxygen delivered from OXY-PFOB-NE across human epidermis, we evaluated the effects of combining LMWP-GFs, QCN-NE, and OXY-PFOB-NE on proliferation of keratinocytes and fibroblasts, and the chronic wound closure rate of a diabetic mouse model.Results: The optimal ratios of LMWP-EGF, LMWP-IGF-I, LMWP-PDGF-A, LMWP-bFGF, QCN-NE, and OXY-PFOB-NE were 1, 1, 0.02, 0.02, 0.2, and 60, respectively. Moreover, a Carbopol hydrogel containing LMWP-GFs, QCN-NE, and OXY-PFOB-NE (LMWP-GFs/QCN-NE/OXY-PFOB-NE-GEL) significantly improved scratch-wound recovery of keratinocytes and fibroblasts in vitro compared to that afforded by hydrogels containing each component alone. LMWP-GFs/QCN-NE/OXY-PFOB-NE-GEL significantly accelerated wound-healing in a diabetic mouse model, decreasing wound size by 54 and 35% compared to the vehicle and LMWP-GFs, respectively.Conclusion: LMWP-GFs/QCN-NE/OXY-PFOB-NE-GEL synergistically accelerated the healing of chronic wounds, exerting both rapid and prolonged effects.Keywords: diabetic wound-healing, topical hydrogel, highly skin-permeable growth factors, quercetin, 1-bromoperfluorooctane, oxygen delivery

Oxidized low-density lipoprotein (ox-LDL) leads to atherosclerosis via lectin-like oxidized lipoprotein receptor-1 (LOX-1), one of the major receptor for ox-LDL. Inhibition of the binding of ox-LDL to LOX-1 decreases the proinflammatory and atherosclerotic events. The aim of the present study was to investigate whether protamine, a polybasic nuclear protein, interferes the binding of ox-LDL to LOX-1. Using sandwich ELISA with newly generated antibody, we measured the blocking effect of protamine on the binding of ox-LDL to LOX-1. Protamine dose-dependently inhibited the binding of ox-LDL to LOX-1. DiI-labeled ox-LDL uptake assay in two types of cultured human endothelial cells was performed with fluorescence microplate reader. Activation of extracellular-signal-regulated kinase (ERK)1/2 by ox-LDL was analyzed by immunoblotting. We found that protamine suppressed uptake of ox-LDL in endothelial cells and inhibited ERK1/2 activation by ox-LDL. These results suggest that protamine may possess anti-atherogenic potential by inhibiting ox-LDL binding to LOX-1 through electrostatic interactions.

The proper transition of histones to protamine during spermiogenesis is critical for male fertility. This study aimed to quantify the levels of histones and protamines mRNA in sperms of infertile couples and their possible effect on the sperm's quality and chromatin integrity. Spermatozoa from 53 normal and 75 patients were enrolled in this study. Histones and Protamine mRNAs were extracted, reverse-transcribed and applied to real-time quantitative PCR. Chromomycin A3 staining was used to assess protamination and chromatin integrity, and Eosin-Nigrosine-Test and HOS-Test was used to evaluate the sperm's vitality and membrane integrity respectively. Levels of histones H2A and H2B mRNA in patient's sperms were significantly (p 0.01) higher than that of normal (31.22 ± 2.91, 30.03 ± 2.05 vs. 25.62 ± 1.98, 27.23 ± 3.04, respectively). Protamine PRM2 mRNA in patient's sperms (20.55 ± 2.01) was significantly lower than in normal (21.73 ± 2.64, p 0.01). The PRM1/PRM2 and H2A/H2B mRNAs ratios were significantly higher (p 0.01) in patients than normal (1.02 ± 0.10, 1.04 ± 0.07 vs. 0.98 ± 0.06, 0.94 ± 0.08 respectively). Also, the sperm's nuclear histones to protamines transcripts ((H2A + H2B)/(PRM1 + PRM2)) ratios of patients (1.49 ± 0.16) was significantly higher (p 0.01) than that of normal (1.25 ± 0.15). Histone/protamine transcripts [((H2A + H2B)/(PRM1 + PRM2)) mRNAs ratios] were negatively correlated (p 0.05) with sperm's count, total count, motility, progressive motility, normal morphology, membrane integrity and positively with chromatin decondensation. The data suggests that histones/protamines mRNAs ratios are important for a sperm's quality and therefore could be used as predictors for male infertility. Also, validation study may be required to confirm the study conclusion.

Protamine and heparin are the most important polyionic drugs used during surgeries and extracorporeal therapies. In this article, a selective and sensitive fluorescence method for the detection of both protamine and heparin was developed by using bovine serum albumin stabilised copper nanoclusters. Blue emitting fluorescent copper nanoclusters were synthesized in aqueous solution using bovine serum albumin as a capping agent and a reducing agent. A one pot microwave assisted method was adopted to synthesize fluorescent copper nanoclusters showing emission at 410 nm upon excitation at 330 nm. The fluorescence of copper nanoclusters was found to be enhanced after the addition of protamine and the limit of detection obtained is 0.12 ng mL-1. The significant enhancement in fluorescence can be attributed to the electrostatic interactions between the copper nanocluster and protamine. In contrast, the enhanced fluorescence intensity of the copper nanocluster with protamine added was decreased after the addition of heparin, and the copper nanocluster regained its original fluorescence intensity. This can be attributed to the strong interaction of protamine with heparin and the limit of detection was calculated as 0.0406 ng mL-1. The selectivity and sensitivity of the sensor for both protamine and heparin were also determined in the presence of potentially co-existing biomolecules, cations, and anions and satisfactory results were obtained. Additionally the validity of the proposed protamine and heparin sensor was attested in real sample matrices such as human urine samples and human blood serum samples. The results exhibited that the recovery percentage of protamine and heparin reached 98-99% and 92-99% in urine samples and 97-99% in serum samples.