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Insects are the largest group of animals when it comes to the number and diversity of species. Yet, with the exception of Drosophila, no information is currently available on the primary structure of their sperm nuclear basic proteins (SNBPs). This paper represents the first attempt in this regard and provides information about six species of Neoptera: Poecillimon thessalicus, Graptosaltria nigrofuscata, Apis mellifera, Nasonia vitripennis, Parachauliodes continentalis, and Tribolium castaneum. The SNBPs of these species were characterized by acetic acid urea gel electrophoresis (AU-PAGE) and high-performance liquid chromatography fractionated. Protein sequencing was obtained using a combination of mass spectrometry sequencing, Edman N-terminal degradation sequencing and genome mining. While the SNBPs of several of these species exhibit a canonical arginine-rich protamine nature, a few of them exhibit a protamine-like composition. They appear to be the products of extensive cleavage processing from a precursor protein which are sometimes further processed by other post-translational modifications that are likely involved in the chromatin transitions observed during spermiogenesis in these organisms. [ABSTRACT FROM AUTHOR]

Objectives: In our center, an unusual rate of patients had abnormalities of hemostasis in immediate postoperative period of cardiac surgery. Our objectives were to identify the cause of these sudden hemostasis abnormalities and to evaluate the performances of point of care coagulation testing. Methods: In this prospective and descriptive study, we included 33 consecutive patients undergoing elective cardiac surgery for 1 month. Heparin-induced anticoagulation and calculation of the protamine dose were tested by the Hemostasis Management System Plus device (Medtronic, Minneapolis, MN, USA). Fifteen minutes after the end of the protamine infusion, activated clotting time (ACT), activated partial thromboplastin time and anti Xa activity were measured. In case of unusual clinical bleeding, a Quantra analysis (Stago, HemoSonics LLC, Charlottesville, VA) was added. Results: Residual antiXa activity >0.2 IU/mL after neutralization was present in 44% of patients. Our investigation concluded incomplete heparin reversal. There was no association between cellular reinfusate and the presence of heparin. The unusual rate of hemostasis abnormalities was explained by a less efficient protamine reversal of heparin. ACT and Clot Time Ratio (CTR, Quantra system) correlated with AntiXa with Spearman's coefficients of 0.85 (p <.0001) and 0.95 (p =.0012), respectively. About ACT, a threshold of 150 seconds had a sensitivity of 85% [58–97] and a specificity of 85% [58–97%] for detection of AntiXa>0.2. For CTR, a threshold of 1.4 had a sensitivity of 67% [30–94] and a specificity of 100% [18–100]. Conclusion: The use of point of care coagulation testing is effective in detecting incomplete reversal of heparin. [ABSTRACT FROM AUTHOR]

• BDE-209 suppressed meiotic promoter expression by suppressing Sohlh1 expression. • BDE-209 inhibited the chromosomal synaptonemal complex formation by L3MBTL2. • BDE-209 inhibited histone ubiquitination by L3MBTL2 affecting spermatid into sperm. • L3MBTL2 regulated spermatogenesis by affecting meiosis and spermatid deformation. The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Melatonin (MEL) is an indole amine molecule primarily produced in the pineal gland. Melatonin has been shown in numerous studies to have antifibrotic effects on the kidney, liver, and other organs. However, it is still unclear how melatonin works in bladder fibrosis. We explored how melatonin affects animals with bladder fibrosis and the underlying mechanisms. Materials and methods: MEL was used to treat human bladder smooth muscle cells (HBdSMCs) after they were stimulated with transforming growth factor-β1 (TGF-β1) in vitro. Proteomic analysis and bioinformatic analysis of the altered expression of these proteins were subsequently performed on HBdSMCs from the different processing methods. To construct an in vivo bladder fibrosis model, we injected protamine sulfate (PS) and lipopolysaccharide (LPS) twice a week into the rat bladder for six weeks. After two weeks of PS/LPS treatment, the mice in the treatment group were treated with MEL (20 mg/kg/d) for 4 weeks. Finally, we detected the expression of fibrosis markers from different perspectives. The TGF-β1/Smad pathway and epithelial–mesenchymal transition (EMT) in cell and bladder tissues were also identified. Further proteomic analysis was also performed. Results: In vitro, we found that TGF-β1 treatment enhanced the expression of the fibrosis markers collagen III and α-SMA in HBdSMCs. E-cadherin expression decreased while the TGF-β1/Smad pathway was activated. Vimentin and N-cadherin expression was also elevated at the same time. Similar findings were observed in the LPS group. After MEL treatment, the expression of collagen III and α-SMA decreased, the expression of E-cadherin increased, and the expression of vimentin and N-cadherin also decreased. According to our quantitative proteomics analysis, CCN1 and SQLE may be important proteins involved in the development of bladder fibrosis. MEL decreased the expression of these genes, leading to the relief of bladder fibrosis. Bioinformatics analysis revealed that the extracellular space structure related to metabolic pathways, actin filament binding, and stress fibers can serve as a pivotal focus in the management of fibrosis. Conclusion: Melatonin attenuates bladder fibrosis by blocking the TGF-β1/Smad pathway and EMT. CCN1 appears to be a possible therapeutic target for bladder fibrosis. [ABSTRACT FROM AUTHOR]

This study was designed to investigate the effectiveness of protamine sulfate as an apelin receptor blocker on cholestatic liver fibrosis persuaded by common bile duct ligation (BDL) in experimental rats and to examine some of the related mechanisms. Three groups of adult male Wistar rats were included: control (sham-operated), BDL, and BDL + protamine sulfate groups. All groups were examined after 4 weeks for serum apelin, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), hepatic interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), transforming growth factor-β (TGF-β), and hydroxyproline content, and for extracted[PF1][H2] liver histopathological studies. BDL significantly increased serum apelin, total bilirubin, AST, ALT, ALP, hepatic IL-6, TNF-α, MDA, TGF-β and hydroxyproline content, but it significantly reduced serum albumin level and hepatic GPx and SOD activities. Serum level of apelin significantly revealed positive correlations with TNF-α, MDA, TGF-β, and hydroxyproline content. On the other hand, protamine sulfate significantly attenuated these changes, with no effect on either serum apelin or bilirubin levels, in the treated group. It also improved the hepatic histopathological changes. Protamine sulfate, maybe through its apelin receptor blockade, ameliorated cholestatic liver injury, inflammation, and fibrosis induced by BDL. [ABSTRACT FROM AUTHOR]

Reactive blue 2 (RB2) dye specifically binds to the nuclei of human spermatozoa under weakly alkaline conditions, thereby providing a new method for assessing sperm quality. However, this technique has not yet been applied to other mammalian species, such as well-established rodent models, which would allow evaluation of the male reproductive toxicity of new drug candidates in nonclinical studies. We aimed to evaluate the usefulness of RB2 staining in assessing testicular and epididymal sperm toxicity in mice using a busulfan-induced infertility model. Male C57BL/6J mice were intraperitoneally administered 40 mg/kg of busulfan. After 28 days, the testes and epididymis were collected and stained with RB2 at pH 10. In vitro evaluations were conducted on uncoated glass slides with RB2 mixed with mouse synthetic protamines, protamines extracted from the human spermatozoa or intracellular protein components from somatic cells without protamines. Following peanut agglutinin lectin histochemistry, RB2-positive cells were observed in elongating and elongated spermatids at all stages except for stages IX–XI of the seminiferous epithelium. After busulfan administration, the proportion of RB2-positive germ cells in the seminiferous tubules was significantly decreased, and no RB2-positive spermatozoa were found in the caput epididymis of treated mice. Aggregates were observed in a mixture of RB2 dye (pH 10) and protamines but not in a mixture of intracellular protein components without protamines, and this specificity was lost at a neutral pH. Our study demonstrated that RB2 specifically stains steps 12–16 spermatids, indicating specific binding to the protamines expressed in these spermatids. The RB2 staining technique has potential as a biomarker for male reproductive toxicity, allowing for the rapid visualization of protamination in an animal model commonly used for the evaluation of male reproductive toxicity. [ABSTRACT FROM AUTHOR]

Introduction: Our study aim was to explore how different protamine-heparin ratios impacted enzymatic coagulation and acellular fibrin clot growth in plasma using an in vitro model. We hypothesized that a low protamine-heparin ratio would be associated with superior fibrin clot growth dynamics. Methods: We performed an in vitro study using 15 plasma samples from a commercial supplier. Different protamine-heparin ratios were added to each donor plasma sample: low ratio (0.7–1), traditional ratio (1–1), and high ratio (1.3–1) and clot formation dynamics were evaluated using a Thrombodynamics analyzer. Study outcomes were initial clot growth velocity and clot size at 30 min. Results: Plasma samples treated with a one-to-one protamine-heparin ratio had significantly lower mean initial clot growth velocity compared to samples treated with a low protamine-heparin ratio; mean difference −2.3 μm/min (95% CI = −4.0 to −0.7, p =.004). Plasma samples treated with a one-to-one protamine-heparin ratio also had significantly smaller mean clot size at 30 min compared to samples treated with a low protamine-heparin ratio; mean difference −54.0 μm (95% CI = −107.6 to −0.4, p =.048). There were no significant differences in mean initial clot growth velocity or clot size at 30 min between plasma samples treated with a high protamine-heparin ratio and those treated with a one-to-one or low protamine-heparin ratio (all p >.05). Conclusions: Plasma samples treated with a low protamine-heparin ratio had superior clot growth velocity and larger clot size at 30 min compared to a one-to-one ratio, supporting the notion that a low protamine-heparin ratio may optimize enzymatic coagulation after cardiopulmonary bypass. [ABSTRACT FROM AUTHOR]

Purpose The aim of this article is to describe a case in which protamine was used for a low-molecular-weight heparin (LMWH) overdose and present an up-to-date review of the literature on the management of LMWH overdose in adults. Summary An unintentional administration of enoxaparin 900 mg occurred in a 73-year-old man with coronavirus disease 2019–related pulmonary embolism. Management of the overdose included a protamine bolus followed by an infusion. Anti–factor Xa levels and activated partial thromboplastin time were monitored. Anti–factor Xa levels declined in a linear fashion irrespective of protamine administration. No bleeding or further thrombotic complications occurred in the patient. A review of the literature revealed that the optimal strategy to treat an LMWH overdose is unknown, with treatment of overdoses ranging from clinical observation to aggressive protamine dosing in reported cases. Although protamine effectively neutralizes unfractionated heparin, it is unable to completely reverse LMWH activity and has variable effects on laboratory measures of LMWH anticoagulant activity. Conclusion The current case report provides additional data to previous literature suggesting that protamine may have a limited effect in decreasing anti–factor Xa levels in LMWH overdose. Continued reporting on the management of LMWH overdoses is warranted to clarify the optimal treatment strategy. [ABSTRACT FROM AUTHOR]

Latent autoimmune diabetes in adults (LADA) is a slow progressive autoimmune destruction of pancreatic beta cells. This condition tends to manifest during adulthood, often around 35 years of age. While LADA can initially be managed by oral medications, eventually the patient will require insulin. We report a case of a 34-year-old woman who was initially treated for type 2 diabetes mellitus but was later diagnosed with LADA. [ABSTRACT FROM AUTHOR]

Simple Summary: Percutaneous hepatic melphalan perfusion (chemosaturation) is a treatment option in patients with inoperable liver metastases which is associated with considerable procedural challenges, especially hemodynamic depression, due to a reduced preload and impaired coagulation caused by the use of heparin. Studies on factors that contribute to bleeding complications and a prolonged intensive care unit length of stay are not available. In this retrospective analysis, we found that high perioperative infusion volumes and the omission of heparin reversal with protamine were associated with postoperative bleeding complications, while high infusion volumes also contributed to a length of stay in the intensive care unit of more than one day, which usually is not required. Furthermore, protamine use was not significantly associated with anaphylactic or thromboembolic complications. Our findings suggest a restrictive perioperative infusion regime and support the use of postoperative protamine for heparin reversal in chemosaturation procedures. Percutaneous hepatic melphalan perfusion (chemosaturation) in patients with liver metastases is known to be associated with procedure-related hemodynamic depression and coagulation impairment, which may cause bleeding complications and/or a prolonged intensive care unit length of stay (ICU LOS). We retrospectively analyzed possible predictive factors for bleeding complications and an ICU LOS > 1 d in a cohort of 31 patients undergoing 90 chemosaturation procedures. Using a multivariable mixed-model approach, we identified the amount of perioperative fluid volume (OR 12.0, 95% CI 2.3–60.0, p = 0.003) and protamine (OR 0.065, 95% CI 0.007–0.55, p = 0.012) to be associated with bleeding complications. Furthermore, the amount of perioperative fluid volume was associated with an ICU LOS > 1 d (OR 5.2, 95% CI 1.4–19.0, p = 0.011). Heparin dosage, melphalan dosage, extracorporeal circulation time, and noradrenaline dosage had no significant effects on outcomes. Protamine use was not associated with anaphylactic or thromboembolic complications. Despite the limited sample size, these results suggest a restrictive perioperative fluid regime to be beneficial, and support the use of protamine for heparin reversal after chemosaturation procedures. Further prospective randomized trials are needed to confirm these findings. [ABSTRACT FROM AUTHOR]

Genetic association studies (GAS) may have the capability to probe the genetic susceptibility alleles in many disorders. This systemic review aimed to assess whether an association exists between gene(s)/allelic variant(s), and varicocele-related male infertility (VRMI). This review included 19 GAS that investigated 26 genes in 1,826 men with varicocele compared to 2,070 healthy men, and 263 infertile men without varicocele. These studies focussed on candidate genes and relevant variants, with glutathione S-transferase gene being the most frequently studied (n = 5) followed by the nitric oxide synthase 3 (NOS3) gene (n = 3) and the phosphoprotein tyrosine phosphatase 1 gene (n = 2). In one study the genes for NAD(P)H quinone oxidoreductase 1, sperm protamine, human 8-oxoguanine DNA glycosylase 1, methylenetetrahydrofolate reductase, polymerase gamma, heat shock protein 90, mitochondrial DNA, superoxide dismutase 2, transition nuclear protein 1, and transition nuclear protein 2, were assessed. There is no clear indication that any of these polymorphisms are sturdily associated with VRMI. However, three studies established that the polymorphic genotype (GT + TT) for rs1799983 polymorphism of the NOS3 gene is more frequent in varicocele patients. Further endeavours such as standardising reporting, exploring complementary designs, and the use of GWAS technology are justified to help replicate these early findings. [ABSTRACT FROM AUTHOR]

Introduction: No antidote or established extracorporeal elimination strategy is available for argatroban. Hemadsorption facilitates elimination of smaller drugs. Case Report: A 34-year-old patient underwent urgent heart transplantation. Because of a history of heparin-induced thrombocytopenia, preoperative anticoagulation was performed with argatroban. Despite ceasing of the continuous infusion of argatroban 2 h before surgery, concentration only declined from 0.60 μg/ml to 0.58 μg/ml before surgery, and the activated clotting time (ACT) value shortly was 223 s. Microvascular bleeding had been observed when starting surgery. A CytoSorb® absorption column was integrated into the system of the heparin-anticoagulated cardiopulmonary bypass (CPB) circuit and a flow of 400 mL/min provided during the 2 h of extracorporeal circulation. The argatroban concentration after weaning from CPB was 0.04 μg/ml and satisfying hemostasis had been achieved after protamine administration. Conclusion: Data indicate that the CytoSorb® absorption column might be an effective tool for quick extracorporeal removal of therapeutic concentrations of argatroban. [ABSTRACT FROM AUTHOR]

Background: Unfractionated heparin (UFH) remains a frequently utilized agent in the emergency department (ED) for management of acute venous thromboembolism (VTE). While various protocols of UFH dosing have been proposed for patients with obesity, the optimal dosing and monitoring strategy is unclear. Objective: This study aims to compare the time to the first therapeutic anti-Xa level in obese acute VTE patients following the use of either total body weight (TBW) or adjusted body weight-based (AdjBW) dosing of UFH in the ED, and to analyze the impact of different dosing strategies on patient outcomes. Methods: Inclusion criteria included adult patients with a BMI > 30 kg/m2, and suspected VTE managed with UFH per institutional protocol utilizing a bolus dose followed by maintenance infusion and anti-Xa monitoring. The primary outcome was time to the first therapeutic anti-Xa level in the group dosed per TBW compared with the group dosed per AdjBW. Safety outcomes included incidence of bleeding events, protamine administration, and mortality. Results: There were 32 patients included in the study. Patients dosed per TBW achieved a median time to first therapeutic anti-Xa level of 14.5 hours compared with 15 hours in the AdjBW group (P =.613). The median therapeutic UFH infusion rate was 16 units/kg/hr in the TBW group compared with 13.5 units/kg/hr in the AdjBW group (P <.001). Safety outcomes were not significantly different between groups. Conclusion: Patients presenting to the ED with acute VTE may be managed with UFH using either a TBW or AdjBW dosing strategy. [ABSTRACT FROM AUTHOR]

Background and Purpose: Interstitial cystitis (=painful bladder syndrome) is a chronic bladder syndrome characterised by pelvic and bladder pain, urinary frequency and urgency, and nocturia. Transient receptor potential (TRP) channels are an attractive target in reducing the pain associated with interstitial cystitis. The current study aims to determine the efficacy of combination of TRP vanilloid 1 (TRPV1) and TRP melastatin 8 (TRPM8) channel inhibition in reducing the pain associated with experimental cystitis in guinea pigs. Experimental Approach: A novel animal model of non‐ulcerative interstitial cystitis has been developed using protamine sulfate/zymosan in female guinea pigs. Continuous voiding cystometry was performed in conscious guinea pigs. Ex vivo "close‐to‐target" single unit extracellular recordings were made from fine branches of pelvic nerves entering the guinea pig bladder. Visceromotor responses in vivo were used to determine the effects of TRP channel antagonists on cystitis‐induced bladder hypersensitivity. Key results: Protamine sulfate/zymosan treatment evoked mild inflammation in the bladder and increased micturition frequency in conscious animals. In cystitis, high threshold muscular afferents were sensitised via up‐regulation of TRPV1 channels, high threshold muscular–mucosal afferents were sensitised via TRPM8 channels, and mucosal afferents by both. Visceromotor responses evoked by noxious bladder distension were significantly enhanced in cystitis and were returned to control levels upon administration of combination of low doses of TRPV1 and TRPM8 antagonists. Conclusions and Implications: The data demonstrate the therapeutic promises of combination of TRPV1 and TRPM8 antagonists for the treatment of bladder hypersensitivity in cystitis. [ABSTRACT FROM AUTHOR]

Background: Combinatorial immunotherapy strategies for enhancing the responsiveness of immune system have shown great promise for cancer therapy. Engineered nanoformulation incorporated toll-like receptor (TLR) 9 agonist CpG ODN has shown more positive results in suppressing tumor growth and can significantly enhance other immunotherapy activity with combinatorial effects due to the innate and adaptive immunostimulatory effects of CpG. Results: In the present work, protamine sulfate (PS) and carboxymethyl β-glucan (CMG) were used as nanomaterials to form nanoparticles through a self-assembly approach for CpG ODN encapsulation to generate CpG ODN-loaded nano-adjuvant (CNPs), which was subsequently mixed with the mixture of mouse melanoma-derived antigens of tumor cell lysates (TCL) and neoantigens to develop vaccine for anti-tumor immunotherapy. The obtained results showed that CNPs was able to effectively deliver CpG ODN into murine bone marrow-derived dendritic cells (DC) in vitro, and remarkably stimulate the maturation of DC cells with proinflammatory cytokine secretion. In addition, in vivo analysis showed that CNPs enhanced anti-tumor activity of PD1 antibody and CNPs-adjuvanted vaccine based on the mixture antigens of melanoma TCL and melanoma-specific neoantigen could not only induce anti-melanoma cellular immune responses, but also elicit melanoma specific humoral immune responses, which significantly inhibited xenograft tumor growth. Furthermore, CD16 CAR-T cells were generated by expressing CD16-CAR in CD3+CD8+ murine T cells. Conclusion: Our results eventually showed that anti-melanoma antibodies induced by CNPs-adjuvanted TCL vaccines were able to collaborate with CD16-CAR-T cells to generate an enhanced targeted anti-tumor effects through ADCC (antibody dependent cell cytotoxicity) approach. CD16 CAR-T cells has thus a great potential to be an universal promising strategy targeting on solid tumor synergistic immunotherapy via co-operation with TCL-based vaccine. [ABSTRACT FROM AUTHOR]

Purpose The case of a patient with a massive acute enoxaparin overdose managed with observation and minimal doses of protamine sulfate is reported. Summary Acute enoxaparin overdoses are uncommonly reported and management is widely variable. A 25-year-old man presented to the emergency department (ED) shortly after reporting that he had attempted suicide by injecting himself with 31 syringes of 80 mg of enoxaparin (a total of 2,480 mg) in the abdomen and other areas of his body. The patient also had self-inflicted superficial lacerations of the forearm. Due to concern over suspected compartment syndrome in the forearm, 25 mg of protamine was administered. Approximately 11 hours after reported enoxaparin self-injection, the patient's activated partial thromboplastin time (aPTT) was 206 seconds, prompting administration of an additional 50 mg of protamine. Three hours later, the aPTT had decreased to 79 seconds, then rose over several hours to 127 seconds before gradually declining to normal values. Protamine administration had no appreciable impact on anti–factor Xa activity. The patient did not require any blood products during the hospital admission. There were no further complications, and the patient was discharged to the inpatient psychiatry service on hospital day 8. Conclusion The case highlights the role of protamine as a reversal agent in the management of low-molecular-weight heparin overdoses. The optimal dosing and efficacy of protamine for this indication needs further investigation. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is characterized by lung extracellular matrix (ECM) remodeling that contributes to obstruction. This is driven, in part by extracellular vesicles (EVs) from activated neutrophils (PMNs), which express on their surface an a-1 antitrypsin (AAT) insensitive form of neutrophil elastase (NE). These EVs are predicted to bind to collagen fibers via Mac-1 integrins, during which time NE can enzymatically degrade the collagen. Protamine sulfate (PS), a cationic compound used safely for decades in humans, has been shown, in vitro, to dissociate this NE from the EV surface, rendering it AAT-sensitive. In addition, a nonapeptide inhibitor, MP-9, has been shown to prevent EV association with collagen. We sought to test whether PS, MP-9, or a combination of the two could effectively prevent NEþ EV-driven ECM remodeling in an animal COPD model. EVs were preincubated with PBS, protamine sulfate (25 µM), MP-9 (50 µM), or a combination of PS and MP-9. These were delivered intratracheally to anesthetized female 10- to 12-wk-old A/J mice for a 7-day time period. One group of mice was euthanized and lungs sectioned for morphometry, and the other group was used for live pulmonary function testing. The effect of alveolar destruction by activated neutrophil EVs was abrogated by pretreatment with PS or MP-9. However, in pulmonary function tests, only the PS groups (and combined PS/MP-9 groups) returned pulmonary function to near-control levels. These data presented here offer an insight into the effective use of PS in therapeutic setting for EV-derived alveolar damage. NEW & NOTEWORTHY Protamine sulfate facilitates the removal of neutrophil elastase (NE) from the surface of extracellular vesicles from activated neutrophils. This "free" NE is no longer protected from inhibition by its endogenous anti-protease, a-1-anti-trypsin. This function of protamine sulfate highlights it as a potential therapeutic strategy for COPD, which may attenuate the disease process. [ABSTRACT FROM AUTHOR]

Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. Here, we show that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. We find that modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs (Mst77Y), leading to the mutant’s known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. We further show that modulo regulates these protamine genes at the step of transcript polyadenylation. We conclude that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility. [ABSTRACT FROM AUTHOR]

Pentaamino acid fullerene C60 derivative is a promising nanomaterial, which exhibited antihyperglycemic activity in high-fat diet and streptozotocin-induced diabetic rats. This study investigates the effect of pentaaminoacid C60 derivative (PFD) in rats with metabolic disorders. Rats were assigned to 3 groups (of 10 rats each) as follows: Group 1 (normal control), group 2 included the protamine-sulfate-treated rats (the untreated group of animals with the model metabolic disorder); group 3 (Protamine sulfate + PFD) included the protamine-sulfate-treated model rats that received an intraperitoneal injection of PFD. Metabolic disorder in rats was initiated by protamine sulfate (PS) administration. The PS + PFD group was injected intraperitoneally with PFD solution (3 mg/kg). Protamine sulfate induces biochemical changes (hyperglycemia, hypercholesterolemia, and hypertriglyceridemia) in the blood and morphological lesions in rat liver and pancreas. The potassium salt of fullerenylpenta-N-dihydroxytyrosine in protamine sulfate-induced rats normalized blood glucose level and the serum lipid profile and improved hepatic function markers. Treatment with PFD restored pancreas islets and liver structure of protamine sulfate-induced rats compared to the untreated group. PFD is a promising compound for further study as a drug against metabolic disorders. [ABSTRACT FROM AUTHOR]

Major vascular complications after transcatheter aortic valve replacement (TAVR) are a pertinent issue and associated with increased morbidity and mortality. We herein describe a case of acute limb ischemia following the administration of protamine sulfate (PS) that was administered to mitigate a bleeding complication post-failure of a vascular access closure device. PS should be used cautiously for the prevention or management of bleeding-site complications following TAVR. The patient described in this case has consented to having his case described in this manuscript. • Major vascular complications after TAVR are associated with increased mortality. • Acute limb ischemia followed administration of protamine sulfate (PS). • PS was given to mitigate bleeding post-vascular access closure device failure. • PS should be used cautiously to manage bleeding-site complications post-TAVR. [ABSTRACT FROM AUTHOR]

Insulin is one of the most important drugs in the treatment of diabetes. There is an increasing interest in the oral administration of insulin as it mimics the physiological pathway and potentially reduces the side effects associated with subcutaneous injection. Therefore, insulin-loaded polyelectrolyte complex (PEC) nanoparticles were prepared by the ionic cross-linking method using protamine sulfate as the polycationic and sodium alginate as the anionic polymer. Taguchi experimental design was used for the optimization of nanoparticles by varying the concentration of sodium alginate, the mass ratio of sodium alginate to protamine, and the amount of insulin. The optimized nanoparticle formulation was used for further in vitro characterization. Then, insulin-loaded PEC nanoparticles were placed in hard gelatin capsules and the capsules were enteric-coated by Eudragit L100-55 (PEC-eCAPs). Hypoglycemic effects PEC-eCAPs were determined in vivo by oral administration to diabetic rats. Furthermore, in vivo distribution of PEC nanoparticles was evaluated by fluorescein isothiocyanate (FITC) labelled nanoparticles. The experimental design led to nanoparticles with a size of 194.4 nm and a polydispersity index (PDI) of 0.31. The encapsulation efficiency (EE) was calculated as 95.96%. In vivo studies showed that PEC-eCAPs significantly reduced the blood glucose level of rats at the 8th hour compared to oral insulin solution. It was concluded that PEC nanoparticles loaded into enteric-coated hard gelatin capsules provide a promising delivery system for the oral administration of insulin. [Display omitted] [ABSTRACT FROM AUTHOR]

Introduction: Pericardial bleeding is a rare but life‐threatening complication of atrial fibrillation (AF) ablation. Patients taking uninterrupted oral anticoagulation (AC) may be at increased risk for refractory bleeding despite pericardiocentesis and administration of protamine. In such cases, andexanet alfa can be given to reverse rivaroxaban or apixaban. In this study, we aim to describe the rate of acute hemostasis and thromboembolic complications with andexanet for refractory pericardial bleeding during AF ablation. Methods and Results: In this multicenter, case series, participating centers identified patients who received a dose of apixaban or rivaroxaban within 24 h of AF ablation, developed refractory pericardial bleeding during the procedure despite pericardiocentesis and administration of protamine and received andexanet. Eleven patients met inclusion criteria, with mean age of 73.5 ± 5.3 years and median CHA2DS2‐VASc score 4 [3–5]. All patients received protamine and pericardiocentesis, and 9 (82%) received blood products. All patients received a bolus of andexanet followed, in all but one, by a 2‐h infusion. Acute hemostasis was achieved in eight patients (73%) while three required emergent surgery. One patient (9%) experienced acute ST‐elevation myocardial infarction after receiving andexanet. Therapeutic AC was restarted after a mean of 2.2 ± 1.9 days and oral AC was restarted after a mean of 2.9 ± 1.6 days, with no recurrent bleeding. Conclusion: In patients on uninterrupted apixaban or rivaroxaban, who develop refractory pericardial bleeding during AF ablation, andexanet can achieve hemostasis thereby avoiding the need for emergent surgery. However, there is a risk of thromboembolism following administration. [ABSTRACT FROM AUTHOR]

Simple Summary: The most frequent cancer in women is breast cancer (BC), which presents a significant risk to their good health. Over the past few decades, several factors have significantly affected BC progression and treatment. Additionally, microRNAs (miRNAs) that govern several oncogenes and tumor suppressors significantly control the expression networks during BC progression. As a result, miRNA-based therapies that changes these networks may modify the cellular activity to the point where they can treat BC. However, the most substantial challenges in developing such miRNA therapies are the stability and efficacy of their delivery systems. A comprehensive update describing various tumor suppressor miRNAs (TS miRNAs) in BC and their various delivery systems are discussed in this review. The death rate from breast cancer (BC) has dropped due to early detection and sophisticated therapeutic options, yet drug resistance and relapse remain barriers to effective, systematic treatment. Multiple mechanisms underlying miRNAs appear crucial in practically every aspect of cancer progression, including carcinogenesis, metastasis, and drug resistance, as evidenced by the elucidation of drug resistance. Non-coding RNAs called microRNAs (miRNAs) attach to complementary messenger RNAs and degrade them to inhibit the expression and translation to proteins. Evidence suggests that miRNAs play a vital role in developing numerous diseases, including cancer. They affect genes critical for cellular differentiation, proliferation, apoptosis, and metabolism. Recently studies have demonstrated that miRNAs serve as valuable biomarkers for BC. The contrast in the expression of miRNAs in normal tissue cells and tumors suggest that miRNAs are involved in breast cancer. The important aspect behind cancer etiology is the deregulation of miRNAs that can specifically influence cellular physiology. The main objective of this review is to emphasize the role and therapeutic capacity of tumor suppressor miRNAs in BC and the advancement in the delivery system that can deliver miRNAs specifically to cancerous cells. Various approaches are used to deliver these miRNAs to the cancer cells with the help of carrier molecules, like nanoparticles, poly D, L-lactic-co-glycolic acid (PLGA) particles, PEI polymers, modified extracellular vesicles, dendrimers, and liposomes. Additionally, we discuss advanced strategies of TS miRNA delivery techniques such as viral delivery, self-assembled RNA-triple-helix hydrogel drug delivery systems, and hyaluronic acid/protamine sulfate inter-polyelectrolyte complexes. Subsequently, we discuss challenges and prospects on TS miRNA therapeutic delivery in BC management so that miRNAs will become a routine technique in developing individualized patient profiles. [ABSTRACT FROM AUTHOR]

Introduction: Carotid endarterectomy (CEA) for symptomatic stenosis reduces further stroke risk. Post-CEA haematoma increases the risk of complications including stroke. There are few studies considering protocols aimed at reducing post-CEA haematoma rates. Presented are the outcomes of a protocol developed to reduce this surgical complication. Method: The protocol was implemented in 112 consecutive CEA. It involves stepwise additional measures to ensure haemostasis before wound closure. Attention to bleeding points is followed by light compression for 10 min. Protamine is then given if haemostasis has not been achieved. If after 20 min the problem persists Tranexamic acid is given. Following a further 20 min if haemostasis is not yet achieved a platelet transfusion is undertaken. Haematoma rates, return to theatre for post-operative haematoma and other complications were compared with 100 consecutive pre-protocol introduction CEA cases. Results: Of 112 CEA patients, 19 received protamine, 8 protamine and tranexamic acid. One case required platelet transfusion. Neck haematoma rate fell from 10 to 3 cases (P =.02, OR: 0.25 [95% CI.07-.94]), of which returned to theatre for haematoma evacuation fell from 6 to 1 case (P =.03, OR: 0.14 [95% CI.02-1.19]). 30 day stroke and death rate reduced from 5% to 1.8% (P =.11, OR: 0.35 [95% CI.07-1.82]). Conclusion: The stepwise haemostasis intraoperative protocol can reduce post-CEA haematoma rates. [ABSTRACT FROM AUTHOR]

Background: Increased sperm DNA damage is known as one of the causes of recurrent pregnancy loss (RPL) which can be due to increased levels of oxidative stress. Therefore, the aim of this study was to assess the effect of alphalipoic acid (ALA) on sperm parameters and sperm functions in couples with a history of RPL. Materials and Methods: In this post hoc analysis in clinical trial study, a total of 37 couples with RPL (n=12 and n=25 for placebo and ALA groups, respectively) were considered. Men were treated with ALA (600 mg/day) or placebo for 80 days. Semen samples were acquired from the participants before initiation and after completion of the medication course and assessed regarding conventional sperm parameters, chromatin damage/integrity, intracellular oxidative stress, lipid peroxidation, and seminal antioxidant characteristics. Individuals were further followed up for twelve months for pregnancy occurrence and outcomes. Finally, after excluding patients with no history of RPL, the data was analyzed. Results: No significant differences were observed between the baseline measures of the aforementioned parameters except for seminal volume. After the intervention, the mean sperm DNA damage, protamine deficiency, and persisted histones were significantly lower in the ALA group than in placebo receivers (P<0.05). A decrease in the mean of seminal total antioxidant capacity (P=0.03), malondialdehyde (P=0.02), and sperm DNA damage (P=0.004) as well as an increase in sperm total motility (P=0.04) after treatment with ALA was noticed. In addition, the mean of protamine deficiency and persisted histones were declined post-ALA therapy (P=0.003 and 0.002, respectively). The percentage of spontaneous pregnancy in the ALA group (4 of 25 cases; 16%) was higher than in the placebo group (1 of 12, 8.3%). Conclusion: ALA-therapy attenuates sperm DNA damage and lipid peroxidation while enhancing sperm total motility and chromatin compaction in the male partner of couples with PRL (registration number: IRCT20190406043177N1). [ABSTRACT FROM AUTHOR]

Objectives: In recent years, smoking water pipes or hookah has increased among adolescents in most countries. Although there is evidence in support of the negative effects of this type of smoking on human health, such as the increased risk of lung disease, little is known about the potential effects of hookah smoking on the male reproductive system, especially on the molecular aspects of sperm. Patients and methods: This cross-sectional study examined sperm DNA fragmentation index, protamine 1 and 2 (PRM1 and PRM2) genes expression, and oxidant status in normozoospermic hookah smokers in comparison with non-smoker controls. Results: Our results showed significantly higher rates of DNA fragmentation, protamine deficiency, and abnormal chromatin condensation in the spermatozoa of hookah smokers (P <.0001). Also, protamine gene expression showed a remarkable decrease in hookah smokers (1.55 ± 2.54 and 0.33 ± 0.54) compared to the controls (3.49 ± 5.41 and 1.22 ± 1.96), although the reduction was not statistically significant (P =.155 and P =.066, respectively). Moreover, a significantly higher level of semen MDA was observed in the case group compared to the controls (0.39 ± 1.04 vs 0.15 ± 0.21; P =.013). Conclusion: According to our study, although hookah smoking does not have a significant effect on sperm parameters, it may have deleterious effects on DNA integrity, oxidative status, and nuclear protein levels of spermatozoa. [ABSTRACT FROM AUTHOR]

OBJECTIVES: In recent years, smoking water pipes or hookah has increased among adolescents in most countries. Although there is evidence in support of the negative effects of this type of smoking on human health, such as the increased risk of lung disease, little is known about the potential effects of hookah smoking on the male reproductive system, especially on the molecular aspects of sperm. PATIENTS AND METHODS: This cross-sectional study examined sperm DNA fragmentation index, protamine 1 and 2 (PRM1 and PRM2) genes expression, and oxidant status in normozoospermic hookah smokers in comparison with non-smoker controls. RESULTS: Our results showed significantly higher rates of DNA fragmentation, protamine deficiency, and abnormal chromatin condensation in the spermatozoa of hookah smokers (P < .0001). Also, protamine gene expression showed a remarkable decrease in hookah smokers (1.55 ± 2.54 and 0.33 ± 0.54) compared to the controls (3.49 ± 5.41 and 1.22 ± 1.96), although the reduction was not statistically significant (P= .155 and P = .066, respectively). Moreover, a significantly higher level of semen MDA was observed in the case group compared to the controls (0.39 ± 1.04 vs 0.15 ± 0.21; P = .013). CONCLUSION: According to our study, although hookah smoking does not have a significant effect on sperm parameters, it may have deleterious effects on DNA integrity, oxidative status, and nuclear protein levels of spermatozoa. [ABSTRACT FROM AUTHOR]

Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34+ hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications. [ABSTRACT FROM AUTHOR]

Tryptase test can be used as a clinical marker of mast cell activation. The present study is was aimed to identify variations in serum tryptase levels and their possible relationships with allergic reactions to protamine in low-risk patients undergoing cardiac bypass surgery. Thirty patients according to American Society of Anesthesiologists physical status III who underwent cardiac bypass surgery were enrolled. This prospective, non-randomised, clinical study was conducted in an operating room. Venous blood samples for tryptase measurements were obtained from cardiac bypass surgery patients upon admission to the operating room and immediately before and 30 min after the initiation of protamine administration. Signs of allergic reactions were recorded and management steps based on rapid effect response-based clinical assessments for diagnosis and treatment decisions during protamine administrations were described. Serum tryptase levels and clinical signs of allergic reactions, primarily mean arterial pressure (MAP), were recorded. Serum tryptase levels increased significantly and progressively during the bypass procedure (study power, 80%; sample size, 28; power of analysis, 99.8% with α=0.05); however, tryptase levels did not reach a sufficiently high level to confirm an allergic reaction. The MAP and heart rate decreased in 50% of the patients. Although tryptase increased significantly when compared with baseline levels, protamine-associated increases were not significant and failed to provide an unequivocal indication of an allergic response to protamine. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate the effectiveness and safety of idarucizumab for periprocedural cardiac tamponade after catheter ablation of atrial fibrillation (AF) in patients treated with dabigatran. Methods: We retrospectively studied 28 patients who received catheter ablation of AF and developed periprocedural cardiac tamponade. Patients were divided into two groups: control group (14 cases) and the study group (14 cases). Patients in the control group were administered warfarin bridged with low molecular weight heparin, while patients in the study group were given dabigatran for anticoagulation. Heparin was used for anticoagulation during surgery in both groups. Patients with cardiac tamponade in control group was reversed with protamine and the ones in study group were given protamine and idarucizumab. In the two groups, operative time, time to resume anticoagulation, bleeding time, length of hospital stay, hemodynamic parameters, coagulation function parameters, number of patients undergoing thoracotomy for hemostasis, pericardiocentesis drainage volume, and pericardial drainage retention time were recorded. Results: There was no statistical difference in operative time and length of hospital stay between the two groups (p > 0.05); however, time to resume anticoagulation and bleeding time were significantly lower in the study group than in the control group, with a statistical difference (p < 0.05). After anticoagulation therapy, there was no apparent change and no statistical difference in the hemodynamic parameters and SaO2 between the two groups (p > 0.05). The pericardial drainage volume retention time was significantly shorter in the study group than in the control group, with a statistical difference (p < 0.05). Conclusion: Idarucizumab can rapidly and effectively reverse the anticoagulant effect of dabigatran in patients with AF who have periprocedural cardiac tamponade after catheter ablation. [ABSTRACT FROM AUTHOR]

Present research aim was to identify functional tests in semen associated with DNA damage and chromatin maturity (protamination) which predict the outcome in assisted reproduction. Couples were grouped according to male partner semen parameters, into normozoospermia (NZs), severe male factor (SMF) and mild male factor (MMF). DNA fragmentation index (DFI) in spermatozoa was analysed by sperms chromatin dispersion (SCD), sperm chromatin structure assay (SCSA) and acridine orange testing (AOT). Chromomycin A3 (CMA3) and toluidine blue (TB) staining to measure sperm chromatin maturity (CM). DFI and chromatin decondensation were significantly lower in N compared to male factor categories (MMF and SMF). Aneuploidy embryos were significantly higher in couples with male factor infertility (MMF and SMF). A positive correlation was observed between fertilization rate (FR) and live birth rate (LBR) with sperm concentration, motility, vitality, normal sperm morphology and negative correlation between sperm DFI and sperm CM. No correlation was observed between embryo aneuploidy and sperm DFI or CM. Lower percentage of spermatozoa chromatin integrity are associated with low fertilization and live birth rate. Male factor infertility, due to impaired semen parameters and chromatin defects could be regarded in future as an indication of IVF/ICSI, and predictor of assisted reproductive techniques outcome. [ABSTRACT FROM AUTHOR]

The present work aimed to evaluate the chromatin compaction of rooster spermatozoa along the male reproductive tract, and to study the vas deferens lining cells, potentially involved in sperm maturation. Chromomycin A3 (CMA3) was used to determine the chromatin compaction of spermatozoa from testis (T), proximal (including epididymis, V1), intermediate (V2) and distal (V3) vas deferens, and ejaculate (E). Six Birchen Leonesa roosters were used. E was obtained in vivo by dorso-ventral massage. V1, V2 and V3 sperm were obtained post mortem (six pairs of vasa deferentia), by flushing. T was obtained by washing the testes, cut in halves. The fixed cells were stained with CMA3 and propidium iodide for flow cytometry assessment. Results showed higher (P < 0.01) median fluorescence intensity (lower chromatin compaction) of T (693.8 ± 30.2) than V1 (546.3 ± 17.7), V2 (515.1 ± 12.1), V3 (517.6 ± 12.3) and E (491.4 ± 16.1). Regarding the percentage of intensely stained cells, T differs (P < 0.05) from V2, V3 and E, V1 differs (P < 0.05) from V3 and E, while V2, V3 and E do not differ. The histological analysis revealed secretory capacity of the vas deferens. Our findings specified that the transit though the vas deferens results in high percentage of compacted chromatin spermatozoa in E. Unlike most mammals, rooster sperm maturation is not completed in the epididymis; it continues along the vas deferens where they obtain fertilising capabilities such as motility. However, neither study has monitored the chromatin compaction (necessary for protecting DNA) of rooster sperm along the vas deferens. We observed that the transit along the vas deferens results in a high percentage of spermatozoa with compacted chromatin; the histological analysis of vas deferens confirmed the presence of secretory cells, possibly related to sperm maturation. [ABSTRACT FROM AUTHOR]

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research. Author summary: Protamines are small sperm-specific proteins crucial to packaging and protecting the paternal genome on its way to the fertilization site. Most mammalian species express only protamine 1. However, primates and rodents additionally express protamine 2. Protamine 2 differs mainly in its N-terminal domain (cP2), which is sequentially cleaved off during paternal chromatin packaging. Alteration in this process has been associated with infertility. However, the precise role of cP2 is still a mystery. We generated cP2 deficient mice and demonstrate, that loss of cP2 results in incomplete histone-to-protamine transition, resulting in sperm DNA degradation and infertility. Evidently, cP2 helps in orchestrating the fine-tuned dynamics of DNA-hypercondensation while protecting DNA integrity and aiding removal of DNA-bound transition proteins. [ABSTRACT FROM AUTHOR]

The objective of the study was to develop PEGylated protamine letrozole nanoparticles to combat human breast cancer by modifying the release pattern of letrozole. Breast cancer is amongst the most prevalent diseases in women due to overactivity of human epidermal growth factor receptor 2 (HER2). PEG-protamine letrozole nanoparticle formulation was designed and optimized to alter the release pattern of the drug. The size, morphology, and structure of PEG-protamine letrozole NP were characterized by FTIR, XRD, Zetasizer, and SEM analysis. The result showed the PEG-protamine letrozole nanoparticles were irregular in shape and have size ranging from 258 nm to 388 nm, polydispersity index 0.114 to 0.45, zeta potential of 11.2 mV, and entrapment efficiency 89.93%. XRD studies have confirmed that the crystal structure of letrozole has become amorphous. The drug release study maintained the prolonged release for 72 hours. Moreover, the PEG-protamine letrozole NPs displayed a strong anticancer action compared to MCF-7 cells with an IC50 70 μM for letrozole and 50 μM for PEG-protamine letrozole NPs. Overall, our results indicate that letrozole PEG-protamine NPs alter the release profile of letrozole, which could be an excellent approach for overcoming letrozole resistance in human breast cancer. [ABSTRACT FROM AUTHOR]

Background: Human sperm chromatin condensation is a sum of epigenetic events that allows for the near-complete replacement of histones with protamines. Under high-magnification microscopy, nuclear vacuoles have been described as thumbprints with poor chromatin condensation. The objective of this study is to examine whether vacuolated spermatozoa carry specific epigenetic marks, which may influence embryo development. Methods: The presence and three-dimensional distribution of ten epigenetic marks (protamine-P2, histone-H3, H3K4me1/me2/me3, H3K9me1/me2/me3, H3K27me3, H4k20me2) were evaluated and compared in morphometrically normal spermatozoa according to the presence or absence of a large vacuole occupying more than 15% of the head surface (n = 4193). Results: Vacuolated spermatozoa were significantly more frequently labelled with H3 and H3K4me3 than normal spermatozoa (88.1% ± 2.7 and 78.5% ± 5.2 vs. 74.8% ± 4.8 and 49.1% ± 7.4, respectively; p = 0.009 and p < 0.001) and significantly less marked by P2 and H3K27me3 (50.2% ± 6.2 and 63.9% ± 6.3 vs. 82.1% ± 4.4 and 73.6% ± 5.1, respectively; p < 0.001 and p = 0.028). In three dimensions, vacuoles are nuclear concavities filled with DNA carrying the H3K4me3 marker. Conclusion: High-magnification microscopy is a simple tool to estimate in real time the sperm epigenetic profile. The selection of normal spermatozoa without vacuoles and the deselection of spermatozoa with vacuoles appear to be epigenetically favorable to embryo development and safe offspring. [ABSTRACT FROM AUTHOR]

One of the most astonishing examples of chromatin remodelling occurs during the maturation of male germ cells, where changes in protein structure, as well as chromatin compaction, take place. During the post-meiotic formation of sperm (spermiogenesis), chromatin appears super-condensed, and transcriptionally inactive, allowing for a more hydrodynamic sperm head, and preventing physical and chemical damage to DNA. DNA is closely condensed in the mature sperm nucleus through its linkage with sperm-specific nuclear proteins called sperm nuclear basic proteins, clustered in three categories: histones, protamines and protamine-like proteins. Fish represent a unique group of vertebrates, including species with specific proteins from each type, such as Sparus aurata, Mullus surmuletus, Dicentrarchus labrax, and Scyliorhinus canicula, which display histones, protamine-like proteins, protamines, and keratinous protamines, respectively. Phylogenetically, these proteins are evolutionarily related, presenting a sporadic and non-random distribution, as a result of vertical evolution, where only histones would be found in more primitive species, and protamines would be restricted to those species located at the uppermost branches of the phylogenetic tree. The relative frequency of this transition is almost insignificant during the differentiation of genera, and species, and very small amongst different families, but, very noticeable amongst different orders. Thus, the aim of this study is to gather the existing background related to sperm nuclear basic proteins in fish, showing a general perspective of the state of the art about diversity, and nuclear reorganization of sperm chromatin during spermiogenesis, and the evolution and phylogenetic distribution of these proteins in fish sperm. [ABSTRACT FROM AUTHOR]

SMYD5 belongs to a special class of protein lysine methyltransferases with an MYND (Myeloid-Nervy-DEAF1) domain inserted into a SET (Suppressor of variegation, Enhancer of Zeste, Trithorax) domain. Despite recent advances in its functional characterization, the lack of the crystal structure has hindered our understanding of the structure-and-function relationships of this most unique member of the SMYD protein family. Here, we demonstrate the reliability of using AlphaFold structures for understanding the structure and function of SMYD5 by comparing the AlphaFold structures to the known crystal structures of SMYD proteins, using an inter-residue distance maps-based metric. We found that the AlphaFold confidence scores are inversely associated with the refined B-factors and can serve as a structural indicator of conformational flexibility. We also found that the N-terminal sequence of SMYD5, predicted to be a mitochondrial targeting signal, contains a novel non-classical nuclear localization signal. This sequence is structurally flexible and does not have a well-defined conformation, which might facilitate its recognition for SMYD5's cytonuclear transport. The structure of SMYD5 is unique in many aspects. The "crab"-like structure with a large negatively charged cleft provides a potential binding site for basic molecules such as protamines. The less positively charged MYND domain is associated with the undetectable DNA-binding ability. The most surprising feature is an incomplete target lysine access channel that lacks the evolutionarily conserved tri-aromatic arrangement, being associated with the low H3/H4 catalytic activity. This study expands our understanding of the SMYD protein family from a classical two-lobed structure to a structure of its own kind, being as a fundamental determinant of its functional divergence. [ABSTRACT FROM AUTHOR]

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies. [ABSTRACT FROM AUTHOR]

Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm chromatin protamination in three subpopulations of fertile normozoospermic controls and infertile patients with oligo-/oligoasthenozoospermia. For the first time, a sequential staining protocol was applied, which allowed researchers to analyse 5mC/5hmC levels by immunofluorescence staining, with a previously determined chromatin protamination status (aniline blue staining), using the same spermatozoa. TUNEL assay determined the sperm DNA fragmentation level. The 5mC/5hmC levels were diversified with respect to chromatin protamination status in both studied groups of males, with the highest values observed in protaminated spermatozoa. The linkage between chromatin protamination and 5mC/5hmC levels in control males disappeared in patients with deteriorated semen parameters. A relationship between 5mC/5hmC and sperm motility/morphology was identified in the patient group. Measuring the 5mC/5hmC status of sperm DNA according to sperm chromatin integrity provides evidence of correct spermatogenesis, and its disruption may represent a prognostic marker for reproductive failure. [ABSTRACT FROM AUTHOR]

Simple Summary: The functional characterization of the BRD4-NUT fusion protein as the driver of the highly aggressive NUT Carcinoma is fundamental to the understanding of the mechanisms responsible for the genome-wide hyperacetylation of histones prior to their eviction during the final stages of sperm cells maturation. In maturing sperm cells, a major genome re-organization takes place, which includes a global increase in the acetylation of histones prior to their replacement by protamines, the latter being responsible for the tight packaging of the male genome. Understanding the function of the oncogenic BRD4-NUT fusion protein in NUT carcinoma (NC) cells has proven to be essential in uncovering the mechanisms underlying histone hyperacetylation in spermatogenic cells. Indeed, these studies have revealed the mechanism by which a cooperation between BRD4, a bromodomain factor of the BET family, NUT, a normally testis-specific factor, and the histone acetyltransferase p300, induces the generation of hyperacetylated chromatin domains which are present in NC cells. The generation of Nut ko mice enabled us to demonstrate a genetic interaction between Nut and Brdt, encoding BRDT, a testis-specific BRD4-like factor. Indeed, in spermatogenic cells, NUT and p300 interact, which results in an increased acetylation of histone H4 at both positions K5 and K8. These two positions, when both acetylated, are specifically recognized by the first bromodomain of BRDT, which then mediates the removal of histone and their replacement by protamines. Taken together, these investigations show that the fusion of NUT to BRD4 in NUT Carcinoma cells reconstitutes, in somatic cells, a functional loop, which normally drives histone hyperacetylation and chromatin binding by a BET factor in spermatogenic cells. [ABSTRACT FROM AUTHOR]

Chromatin remodelling in spermatids is an essential step in spermiogenesis and involves the exchange of most histones by protamines, which drives chromatin condensation in late spermatids. The gene Rimklb encodes a citrylglutamate synthase highly expressed in testes of vertebrates and the increase of its reaction product, ß-citrylglutamate, correlates in time with the appearance of spermatids. Here we show that deficiency in a functional Rimklb gene leads to male subfertility, which could be partially rescued by in vitro fertilization. Rimklb-deficient mice are impaired in a late step of spermiogenesis and produce spermatozoa with abnormally shaped heads and nuclei. Sperm chromatin in Rimklb-deficient mice was less condensed and showed impaired histone to protamine exchange and retained transition protein 2. These observations suggest that citrylglutamate synthase, probably via its reaction product ß-citrylglutamate, is essential for efficient chromatin remodelling during spermiogenesis and may be a possible candidate gene for male subfertility or infertility in humans. [ABSTRACT FROM AUTHOR]

Background: Heparin diminishes thrombin generation (TG) because it decreases the survival time of thrombin in plasma. Under heparin therapy, the TG curve therefore does not reflect the true hemostatic status of the patient. Aim: We investigated how far the in vitro addition of a heparin antagonist can restore the underlying TG capacity. Materials & Methods: Five different heparin antagonists were tested: polybrene, protamine sulfate, heparinase type 1, heparinase HEP‐TEM, and (Z‐GGR)2‐rhodamine (P2Rho). Results and Conclusion: Polybrene, P2Rho, and heparinase HEP‐TEM effectively neutralized heparin at prophylactic and therapeutical doses of both low molecular weight and unfractionated heparin. The advantages and limits of each molecule and the most favorable combinations of TG‐trigger and antagonist are discussed. [ABSTRACT FROM AUTHOR]

Diabetes negatively affects the reproductive system. This present study investigated the effects of aerobic training on protamine 1 and 2 mRNA expression, sex hormones, antioxidant defence and sperm quality in diabetic rats. Thirty‐six male Wistar rats were randomly allocated into three groups including diabetic training (DT) group, diabetic (D) group and control (C) group. Rats in DT were exercised 5 times per week for 8 weeks. Blood samples were collected for evaluation of sex hormones 48 h after the last training session. Also, the testes were removed and subjected to histological evaluation and semen analysis. Testicular mRNA expressions of protamines were determined by RT‐qPCR. Protamines 1 and 2, and the ratio of protamine 1 to protamine 2 were significantly lower in DT and D groups compared with C group (p < 0.01). LH and testosterone levels were significantly lower in D group compared with DT and C group (p < 0.01). Malondialdehyde was significantly lower in DT and C groups compared with D group (p < 0.001). Sperm parameters were significantly lower in D group compared with C group (p < 0.01). Our findings suggest that aerobic training may mitigate the negative impact of diabetes on sex hormones, oxidative stress, protamine content and sperm parameters in male rats. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial blight, is one of the major threats to rice productivity. Yet, the molecular mechanism of rice- Xoo interaction is elusive. Here, we report comparative proteome profiles of Xoo susceptible (Dongjin) and resistant (Hwayeong) cultivars of rice in response to two-time points (3 and 6 days) of Xoo infection. Low-abundance proteins were enriched using a protamine sulfate (PS) precipitation method and isolated proteins were quantified by a label-free quantitative analysis, leading to the identification of 3846 proteins. Of these, 1128 proteins were significantly changed between mock and Xoo infected plants of Dongjin and Hwayeong cultivars. Based on the abundance pattern and functions of the identified proteins, a total of 23 candidate proteins were shortlisted that potentially participate in plant defense against Xoo in the resistant cultivar. Of these candidate proteins, a mitochondrial arginase-1 showed Hwayeong specific abundance and was significantly accumulated following Xoo inoculation. Overexpression of arginase 1 (OsArg 1) in susceptible rice cultivar (Dongjin) resulted in enhanced tolerance against Xoo as compared to the wild-type. In addition, expression analysis of defense-related genes encoding PR1, glucanase I, and chitinase II by qRT-PCR showed their enhanced expression in the overexpression lines as compared to wild-type. Taken together, our results uncover the proteome changes in the rice cultivars and highlight the functions of OsARG1 in plant defense against Xoo. [Display omitted] • Low-abundance proteome of Xoo -susceptible and resistant rice cultivars was performed. • Label-free quantitative proteome analysis identified 3846 protein groups. • Proteomics data showed >87% reproducibility and <13% coefficient of variation (CV) values. • Replicates showed Pearson correlation coefficient values 0.948 (Dongjin) and 0.968 (Hwayeong). • An arginase-1 (OsArg1) was significantly accumulated following Xoo inoculation. • Overexpression of OsArg1 resulted in enhanced resistance against Xoo. • Overexpression lines showed enhanced expression of PR1, glucanase I, and chitinase. [ABSTRACT FROM AUTHOR]

Objective: It is necessary to evaluate fertility effective agents to predict assisted reproduction outcomes. This study was designed to examine sperm vacuole characteristics, and its association with sperm chromatin status and protamine-1 (PRM1) to protamine-2 (PRM2) ratio, to predict assisted pregnancy outcomes. Materials and Methods: In this experimental study, ninety eight semen samples from infertile men were classified based on Vanderzwalmen’s criteria as follows: grade I: no vacuoles; grade II: ≤2 small vacuoles; grade III: ≥1 large vacuole and grade IV: large vacuole with other abnormalities. The location, frequency and size of vacuoles were assessed using high magnification, a deep learning algorithm, and scanning electron microscopy (SEM). The chromatin integrity, condensation, viability and acrosome integrity, and protamination status were evaluated for vacuolated samples by toluidine blue (TB) staining, aniline blue, triple staining, and CMA3 staining, respectively. Also, Protamine-1 and protamine-2 genes expression was analysed by reverse transcription-quantitative polymerase chain reaction (PCR). The assisted reproduction outcomes were also followed for each cycle. Results: The results show a significant correlation between the vacuole size (III and IV) and abnormal sperm chromatin condensation (P=0.03 and P=0.02, respectively), and also, protamine-deficient (P=0.04 and P=0.03, respectively). The percentage of reacting acrosomes was significantly higher in the grades III and IV spermatozoa in comparison with normal group. The vacuolated spermatozoa with grade IV showed a high protamine mRNA ratio (PRM-2 was underexpressed, P=0.01). In the IVF cycles, we observed a negative association between sperm head vacuole and fertilization rate (P=0.01). This negative association was also significantly observed in pregnancy and live birth rate in the groups with grade III and IV (P=0.04 and P=0.03, respectively). Conclusion: The results of our study highlight the importance sperm parameters such as sperm head vacuole characteristics, particularly those parameters with the potency of reflecting protamine-deficiency and in vitro fertilization/ intracytoplasmic sperm injection (IVF/ICSI) outcomes predicting. [ABSTRACT FROM AUTHOR]