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Nanoscale Advances Sep 2023Protamine, a small, strongly positively-charged protein, plays a key role in achieving chromatin condensation inside sperm cells and is also involved in the formulation...
Protamine, a small, strongly positively-charged protein, plays a key role in achieving chromatin condensation inside sperm cells and is also involved in the formulation of nanoparticles for gene therapy and packaging of mRNA-based vaccines against viral infection and cancer. The detailed mechanisms of such condensations are still poorly understood especially under low salt conditions where electrostatic interaction predominates. Our previous study, with a refined coarse-grained model in full consideration of the long-range electrostatic interactions, has demonstrated the crucial role of electrostatic interaction in protamine-controlled reversible DNA condensation. Therefore, we herein pay our attention only to the electrostatic interaction and devise a coarser-grained bead-spring model representing the right linear charge density on protamine and DNA chains but treating other short-range interactions as simply as possible, which would be suitable for real-scale simulations. Effective pair potential calculations and large-scale molecular dynamics simulations using this extremely simple model reproduce the phase behaviour of DNA in a wide range of protamine concentrations under low salt conditions, again revealing the importance of the electrostatic interaction in this process and providing a detailed nanoscale picture of bundle formation mediated by a charge disproportionation mechanism. Our simulations also show that protamine length alters DNA overcharging and in turn redissolution thresholds of DNA condensates, revealing the important role played by entropies and correlated fluctuations of condensing agents and thus offering an additional opportunity to design tailored nanoparticles for gene therapy. The control mechanism of DNA-protamine condensates will also provide a better microscopic picture of biomolecular condensates, , membraneless organelles arising from liquid-liquid phase separation, that are emerging as key principles of intracellular organization. Such condensates controlled by post-translational modification of protamine, in particular phosphorylation, or by variations in protamine length from species to species may also be responsible for the chromatin-nucleoplasm patterning observed during spermatogenesis in several vertebrate and invertebrate species.
PubMed: 37705794
DOI: 10.1039/d2na00847e -
International Journal of Molecular... Sep 2023The signal transducer and activator of transcription 3 (), which regulates multiple oncogenic processes, has been found to be constitutively activated in lymphoma,...
The signal transducer and activator of transcription 3 (), which regulates multiple oncogenic processes, has been found to be constitutively activated in lymphoma, suggesting its potential as a therapeutic target. Here, we constructed an anti-CD19-N-(4-carboxycyclohexylmethyl) maleimide N-hydroxysuccinimide ester (SMCC)-protamine (CSP)- small interfering RNA (siRNA) conjugate and demonstrated that the CSP- siRNA conjugate could specifically bind to normal B cells and A20 lymphoma cells in vitro. It decreased the expression in B cell lymphoma cell lines (A20, SU-DHL-2 and OCI-Ly3), resulting in reduced proliferation of lymphoma cells featured with lower S-phase and higher apoptosis. Using an A20 transplantable lymphoma model, we found that the CSP- siRNA conjugate significantly inhibited tumor growth and weight. Ki-67, p-STAT3, STAT3, and serum IL-6 levels were all significantly reduced in A20-bearing mice treated with CSP- siRNA. These findings indicate that specifically targeting siRNA to B cell lymphoma cell lines can significantly decrease activity and inhibit tumor progression in vitro and in vivo, suggesting its potential utilization for cancer treatment.
Topics: Animals; Mice; Adaptor Proteins, Signal Transducing; Antibodies; B-Lymphocytes; Lymphoma, B-Cell; RNA, Small Interfering; STAT3 Transcription Factor
PubMed: 37686472
DOI: 10.3390/ijms241713666 -
The Journal of Extra-corporeal... Sep 2023Systemic anticoagulation with heparin during cardiopulmonary bypass (CPB) should be neutralized by protamine administration to restore normal hemostasis. Our previous...
BACKGROUND
Systemic anticoagulation with heparin during cardiopulmonary bypass (CPB) should be neutralized by protamine administration to restore normal hemostasis. Our previous study showed the protamine-to-heparin ratio (P-to-H) of 1:1 (1 mg protamine:100 IU circulating heparin; 1.0 Ratio) is likely an overestimation. Thus, we reduced the P-to-H in the HMS Plus Hemostasis Management System to 0.9:1 (0.9 Ratio) for 5 months and then to 0.8:1 (0.8 Ratio). We monitored post-operative (post-op) bleeding in the setting of reduced protamine dose (PD).
METHODS
We performed a retrospective study of 632 patients (209 for the 1.0 Ratio, 211 for 0.9 Ratio, 212 for 0.8 Ratio group) who underwent cardiac surgery to measure the reduction of PD and how it affects 24-hour (24 h) post-op chest tube output. We also analyzed the entire data set to explore whether further reduction of P-to-H is warranted.
RESULTS
While there was no difference in the indexed heparin dose among the three groups, we achieved a significant reduction in the indexed actual protamine dose (APDi) by 24% (0.9 Ratio) and 31% (0.8 Ratio) reductions compared to the 1.0 Ratio group. On average, APDi was 88 ± 22, 67 ± 18, and 61 ± 15 mg/m in the 1.0, 0.9, and 0.8 Ratio groups, respectively. We found no significant difference in 24 h post-op bleeding among the three groups.
CONCLUSION
1.0 Ratio at the completion of CPB is likely an excessive administration of protamine. With the stepwise reduction of PD, we observed no increase in post-op bleeding, which may indicate that no meaningful increase in heparin rebound occurred. In addition, further analysis of the entire data set demonstrates that a 0.75 Ratio is likely sufficient to neutralize the heparin completely.
Topics: Humans; Cardiopulmonary Bypass; Heparin; Retrospective Studies; Postoperative Hemorrhage; Protamines
PubMed: 37682208
DOI: 10.1051/ject/2023026 -
European Journal of Pharmaceutics and... Oct 2023Today, macromolecular compounds such as microRNAs (miRNAs) are becoming more and more widespread as leading therapeutics. However, their application is limited mostly...
Today, macromolecular compounds such as microRNAs (miRNAs) are becoming more and more widespread as leading therapeutics. However, their application is limited mostly due to their poor stability, limited cellular uptake, and poor target specificity. Cell-penetrating peptides (CPPs), a group of positively charged peptides, represent a breakthrough as delivery systems for macromolecules. In the present study, we used two types of nanoparticles which differ in the type of CPP used for their manufacturing. The first type is composed of protamine, an arginine rich CPP, which is highly positively charged. The arginine residues are able to form electrostatic interactions with miRNAs, stabilize them, and deliver them to cells. The second type is composed of the N-Ter peptide (also known as MPG), an amphipathic peptide rich in lysine. The positively charged parts of the N-Ter peptide electrostatically stabilize miRNAs, whereas its amphipathic character allows it to successfully traverse cell membranes. We used miRNA-27a, a negative regulator of adipogenesis, to form nanoparticles with the peptides and traced their uptake in 3T3-L1 preadipocytes. Motivated by the lengthy discourse regarding the uptake mechanism of CPPs, the focus of our study was to analyse and understand the internalization of proticles (protamine nanoparticles) and N-Ter complexes. The nanoparticles were characterized regarding size, size distribution, and zeta potential, and their cytotoxicity was tested in 3T3-L1 cells. The uptake studies were performed by varying the experimental conditions such as time, concentration, and temperature, as well as by applying different inhibitors of endocytosis. Furthermore, we assessed the biological effect of miRNA-27a on the pro-adipogenic machinery. The obtained data have shown that protamine and the N-Ter peptide form positively charged nanoparticles through non-covalent complexation. The uptake of proticles and N-Ter complexes was found to be dependent on time, concentration, and temperature, and different uptake pathways were discovered to be involved in the internalization of the different nanoparticles. Furthermore, both types of nanoparticles induced the anti-adipogenic effect of miRNA-27a, demonstrating that this approach can be used as a novel miRNA replacement therapy in the treatment of obesity and obesity-related disorders.
Topics: MicroRNAs; Cell-Penetrating Peptides; Drug Delivery Systems; Endocytosis; Protamines; Arginine
PubMed: 37666365
DOI: 10.1016/j.ejpb.2023.08.019 -
The Journal of Biological Chemistry Oct 2023DNA in sperm is packed with small, charged proteins termed SNBPs (sperm nuclear basic proteins), including mammalian and Drosophila protamines. During spermiogenesis,...
DNA in sperm is packed with small, charged proteins termed SNBPs (sperm nuclear basic proteins), including mammalian and Drosophila protamines. During spermiogenesis, somatic-type chromatin is taken apart and replaced with sperm chromatin in a multistep process leading to an extraordinary condensation of the genome. During fertilization, the ova face a similarly challenging task of SNBP eviction and reassembly of nucleosome-based chromatin. Despite its importance for the animal life cycle, sperm chromatin metabolism, including the biochemical machinery mediating the mutual replacement of histones and SNBPs, remains poorly studied. In Drosophila, Mst77F is one of the first SNBPs loaded into the spermatid nuclei. It persists in mature spermatozoa and is essential for sperm compaction and male fertility. Here, by using in vitro biochemical assays, we identify chaperones that can mediate the eviction and loading of Mst77F on DNA, thus facilitating the interconversions of chromatin forms in the male gamete. Unlike NAP1 and TAP/p32 chaperones that disassemble Mst77F-DNA complexes, ARTEMIS and APOLLO, orthologs of mammalian importin-4 (IPO4), mediate the deposition of Mst77F on DNA or oligonucleosome templates, accompanied by the dissociation of histone-DNA complexes. In vivo, a mutation of testis-specific Apollo brings about a defect of Mst77F loading, abnormal sperm morphology, and male infertility. We identify IPO4 ortholog APOLLO as a critical component of sperm chromatin assembly apparatus in Drosophila. We discover that in addition to recognized roles in protein traffic, a nuclear transport receptor (IPO4) can function directly in chromatin remodeling as a dual, histone- and SNBP-specific, chaperone.
PubMed: 37660905
DOI: 10.1016/j.jbc.2023.105212 -
Medicine Aug 2023To assess the association between insulin regimens and health-related quality of life (HRQoL) after the introduction of basal insulin (BI) among people with type 2...
To assess the association between insulin regimens and health-related quality of life (HRQoL) after the introduction of basal insulin (BI) among people with type 2 diabetes in real-world clinical settings. 16,339 registered people with diabetes who had inadequate glycaemic control by oral agents initiated BI (either single BI or Basal-bolus) and completed a 6-month follow-up from 209 hospitals were included in the analyses. At the end of the follow-up, the switches of insulin regimens, change of HRQoL (EQ-5D-3L) and their associations were assessed. Initial insulin regimens of single BI and of basal-bolus (BI included Glargine, Detemir, and Neutral Protamine Hagedorn) accounted for 75.6% and 24.4%, respectively. At 6 months, regimens used were BI alone (65.2%), basal-bolus (10.4%), and premixed (6.4%), whereas 17.9% stopped all insulin therapy. The visual analogue scale score increased by 5.46 (P < .001), and the index value increased slightly by 0.02 (P < .001). Univariate analysis showed that people with diabetes taking basal-bolus regimen had the greatest improvement on HRQoL in all dimensions, especially in the reduction of the percentage of Pain/Discomfort (by 10.03%) and Anxiety/Depression (by 11.21%). In multivariable analysis, single BI or premixed insulin at 6 months was associated with more improvement of visual analogue scale score compared with stopping all insulin. Improved HRQoL was observed after initiating BI in people with type 2 diabetes . If the same achievement on HbA1c control can be guaranteed, single BI is preferred to other regimens from the viewpoint of HRQoL. Basal-bolus has the most significant potential to increase HRQoL, however, the people with diabetes characteristics differ from those initiating BI alone. Further longitudinal cohort study with a longer study period might be necessary to evaluate the certain effect.
Topics: Humans; Diabetes Mellitus, Type 2; Longitudinal Studies; Quality of Life; Insulin; Body Fluids
PubMed: 37653806
DOI: 10.1097/MD.0000000000034718 -
The EMBO Journal Oct 2023Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome...
Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unclear. Here, we combined RNA-seq, MNase-seq, ChIP-seq, and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the adenoviral genome is arranged in precisely positioned nucleoprotein particles with nucleosome-like characteristics, that we term adenosomes. We identified 238 adenosomes that are positioned by a DNA sequence code and protect about 60-70 bp of DNA. The incoming adenoviral genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replaces pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites and precedes transcriptional activation. Based on our results, we propose a central role for the viral pVII nucleoprotein architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy.
Topics: Nucleosomes; Transcriptional Activation; Chromatin; DNA; Chromatin Assembly and Disassembly; Adenoviridae
PubMed: 37641864
DOI: 10.15252/embj.2023114162 -
Increased Apoptosis in Subcortical Regions of The Visual Pathway in Offspring Born to Diabetic Rats.Cell Journal Aug 2023Diabetes in pregnancy is a prevalent disease that can affect the central nervous system of the fetus by hyperglycemia. This study aimed to investigate the impact of...
OBJECTIVE
Diabetes in pregnancy is a prevalent disease that can affect the central nervous system of the fetus by hyperglycemia. This study aimed to investigate the impact of maternal diabetes on neuronal apoptosis in the superior colliculus (SC) and the lateral geniculate nucleus (LGN) in male neonates born to diabetic mothers.
MATERIALS AND METHODS
In this experimental study, female adult rats were separated into three groups: control, diabetic (induced using an intraperitoneal injection of streptozotocin), and insulin-treated diabetic [diabetes controlled by subcutaneous neutral protamine hagedorn (NPH)-insulin injection]. Male neonates from each group were euthanized on 0, 7, and 14 postnatal days (P0, P7, and P14, respectively), and apoptotic cells were identified using TUNEL staining.
RESULTS
The numerical density per unit area (NA) of apoptotic cells was significantly higher in SC and the dorsal LGN (dLGN) in neonates born to the diabetic rats compared to the control group at P0, P7, and P14. However, insulin treatment normalized the number of apoptotic cells.
CONCLUSION
This study demonstrated that maternal diabetes increased apoptosis in dLGN and SC of male neonates at P0, P7, and P14.
PubMed: 37641418
DOI: 10.22074/cellj.2023.1989649.1232 -
Scientific Reports Aug 2023Interstitial cystitis (IC) has severe clinical symptoms with unclear mechanism. The continuous inflammatory response of the bladder is the basis of its pathogenesis....
Interstitial cystitis (IC) has severe clinical symptoms with unclear mechanism. The continuous inflammatory response of the bladder is the basis of its pathogenesis. Endoplasmic reticulum stress (ERS) is involved in the regulation and development of various inflammatory diseases. And autophagy plays an important role in IC. In this study, we mainly focus on the therapeutic effect of endoplasmic reticulum stress and autophagy on protamine/lipopolysaccharide-induced interstitial cystitis. Female Sprague-Dawley rats were randomized into three experimental groups as follows: sham controls(N), IC alone, and IC+4-PBA.Rats in group IC received 10 mg/ml PS in the urinary bladder, followed by 2 mg/ml LPS instillation after 30 min, IC+4-PBA group SD rats received 4-PBA solution administered intragastrically once a day for 5 days. ERS biomarker (GRP78), autophagy-related proteins (LC3I/II, and Beclin1), autophagic flux biomarker (P62), inflammatory biomarkers (IL-6, TNF-a, NF-κB), apoptotic biomarkers (Caspase 3, Bax) were highest in the IC group compared to IC+4-PBA group and N group and the biomarkers expression in IC+4-PBA group were lower than in the IC group, anti-apoptotic biomarker (Bcl-2) was highest in the N group compared to the IC group and IC+4-PBA group and lower in the IC group than in the IC+4-PBA group, oxidative stress biomarkers (HO-1, NQO-1) were remarkably lower in the control group than in the IC and IC+4-PBA groups and notably lower in the IC group than in the IC+4-PBA group. The histological score and mast cell count demonstrated most severe in the IC group than those in the IC+4-PBA group. TUNEL assay examined the level of apoptosis in IC group was higher than in the IC+4-PBA group. The bladder micturition function was significantly improved with 4-PBA treatment. 4-PBA inhibits ERS to recover autophagic flux, and then to suppress the bladder oxidative stress, the inflammatory reaction and apoptosis, finally improve the bladder urinary function in Protamine/Lipopolysaccharide (PS/LPS) induced IC.
Topics: Female; Rats; Animals; Cystitis, Interstitial; Lipopolysaccharides; Rats, Sprague-Dawley; Autophagy; Endoplasmic Reticulum Stress
PubMed: 37640742
DOI: 10.1038/s41598-023-38584-x -
Molecular Therapy. Nucleic Acids Sep 2023Heparin-induced thrombocytopenia (HIT) is a complication caused by administration of the anticoagulant heparin. Although the number of patients with HIT has drastically...
Heparin-induced thrombocytopenia (HIT) is a complication caused by administration of the anticoagulant heparin. Although the number of patients with HIT has drastically increased because of coronavirus disease 2019 (COVID-19), the currently used thrombin inhibitors for HIT therapy do not have antidotes to arrest the severe bleeding that occurs as a side effect; therefore, establishment of safer treatments for HIT patients is imperative. Here, we devised a potent thrombin inhibitor based on bivalent aptamers with a higher safety profile via combination with the antidote. Using an anti-thrombin DNA aptamer M08s-1 as a promising anticoagulant, its homodimer and heterodimer with TBA29 linked by a conformationally flexible linker or a rigid duplex linker were designed. The dimerized M08s-1-based aptamers had about 100-fold increased binding affinity to human and mouse thrombin compared with the monomer counterparts. Administration of these bivalent aptamers into mice revealed that the anticoagulant activity of the dimers significantly surpassed that of an approved drug for HIT treatment, argatroban. Moreover, adding protamine sulfate as an antidote against the most potent bivalent aptamer completely suppressed the anticoagulant activity of the dimer. Emerging potent and neutralizable anticoagulant aptamers will be promising candidates for HIT treatment with a higher safety profile.
PubMed: 37621412
DOI: 10.1016/j.omtn.2023.07.038