Multi-epitope protein production and its application in the diagnosis of opisthorchiasis.

BACKGROUND: Opisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis. METHODS: The Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique. RESULTS: The recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively. CONCLUSIONS: The high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.

Acute cardiac tamponade after Endostar treatment of non-small cell lung cancer: A case report.

RATIONALE: Recombinant human endostatin (Endostar) is extensively utilized in China for the clinical management of patients with driver gene-negative non-small cell lung cancer (NSCLC) at stage TNM IV. This report describes the case of a lung cancer patient treated exclusively with Endostar maintenance therapy, who experienced a rapid deterioration in respiratory function. PATIENT CONCERNS: The case involved a patient with a pathologically confirmed squamous cell carcinoma of the left lung, treated in our department. Following 1 month of albumin-bound paclitaxel chemotherapy and localized radiotherapy for the left lung lesion, the patient initiated treatment with a single agent, Endostar 30mg, on October 19, 2021. The medication was administered via intravenous infusion over a 7 days. DIAGNOSIS: On October 23, 2021, the patient exhibited symptoms of chest constriction, discomfort, coughing, and sputum production. By October 28, the patient presented with pronounced dyspnea and respiratory distress. An emergency CT scan detected pericardial tamponade and significant deviations in several blood parameters from pretreatment values. INTERVENTIONS: Percardial puncture and catheter drainage were recommended as therapeutic intervention. OUTCOMES: Considering the patient advanced age, the patient and their family opted to refuse this medical procedure, leading to the patient unfortunate demise on November 2, 2021. LESSONS: Medical professionals should remain vigilant for the potential, albeit rare, risk of Endostar inducing acute pericardial tamponade, a severe and potentially fatal complication.

Cysteine oxidation as a regulatory mechanism of Arabidopsis plastidial NAD-dependent malate dehydrogenase.

Malate dehydrogenases (MDHs) catalyze a reversible NAD(P)-dependent-oxidoreductase reaction that plays an important role in central metabolism and redox homeostasis of plant cells. Recent studies suggest a moonlighting function of plastidial NAD-dependent MDH (plNAD-MDH; EC 1.1.1.37) in plastid biogenesis, independent of its enzyme activity. In this study, redox effects on activity and conformation of recombinant plNAD-MDH from Arabidopsis thaliana were investigated. We show that reduced plNAD-MDH is active while it is inhibited upon oxidation. Interestingly, the presence of its cofactors NAD+ and NADH could prevent oxidative inhibition of plNAD-MDH. In addition, a conformational change upon oxidation could be observed via non-reducing SDS-PAGE. Both effects, its inhibition and conformational change, were reversible by re-reduction. Further investigation of single cysteine substitutions and mass spectrometry revealed that oxidation of plNAD-MDH leads to oxidation of all four cysteine residues. However, cysteine oxidation of C129 leads to inhibition of plNAD-MDH activity and oxidation of C147 induces its conformational change. In contrast, oxidation of C190 and C333 does not affect plNAD-MDH activity or structure. Our results demonstrate that plNAD-MDH activity can be reversibly inhibited, but not inactivated, by cysteine oxidation and might be co-regulated by the availability of its cofactors in vivo.

Synergistic effect of umbilical cord extracellular vesicles and rhBMP-2 to enhance the regeneration of a metaphyseal femoral defect in osteoporotic rats.

BACKGROUND: The aim of this study was to evaluate potential synergistic effects of a single, local application of human umbilical cord MSC-derived sEVs in combination with a low dose of recombinant human rhBMP-2 to promote the regeneration of a metaphyseal femoral defect in an osteoporotic rat model. METHODS: 6 weeks after induction of osteoporosis by bilateral ventral ovariectomy and administration of a special diet, a total of 64 rats underwent a distal femoral metaphyseal osteotomy using a manual Gigli wire saw. Defects were stabilized with an adapted Y-shaped mini-locking plate and were subsequently treated with alginate only, or alginate loaded with hUC-MSC-sEVs (2 × 109), rhBMP-2 (1.5 µg), or a combination of sEVs and rhBMP-2 (n = 16 for each group). 6 weeks post-surgery, femora were evaluated by µCT, descriptive histology, and biomechanical testing. RESULTS: Native radiographs and µCT analysis confirmed superior bony union with callus formation after treatment with hUC-MSC-sEVs in combination with a low dose of rhBMP-2. This finding was further substantiated by histology, showing robust defect consolidation 6 weeks after treatment. Torsion testing of the explanted femora revealed increased stiffness after application of both, rhBMP-2 alone, or in combination with sEVs, whereas torque was only significantly increased after treatment with rhBMP-2 together with sEVs. CONCLUSION: The present study demonstrates that the co-application of hUC-MSC-sEVs can improve the efficacy of rhBMP-2 to promote the regeneration of osteoporotic bone defects.

Heterologous expression of P9 from Akkermansia muciniphila increases the GLP-1 secretion of intestinal L cells.

Glucagon-like peptide-1(GLP-1) is an incretin hormone secreted primarily from the intestinal L-cells in response to meals. GLP-1 is a key regulator of energy metabolism and food intake. It has been proven that P9 protein from A. muciniphila could increase GLP-1 release and improve glucose homeostasis in HFD-induced mice. To obtain an engineered Lactococcus lactis which produced P9 protein, mature polypeptide chain of P9 was codon-optimized, fused with N-terminal signal peptide Usp45, and expressed in L. lactis NZ9000. Heterologous secretion of P9 by recombinant L. lactis NZP9 were successfully detected by SDS-PAGE and western blotting. Notably, the supernatant of L. lactis NZP9 stimulated GLP-1 production of NCI-H716 cells. The relative expression level of GLP-1 biosynthesis gene GCG and PCSK1 were upregulated by 1.63 and 1.53 folds, respectively. To our knowledge, this is the first report on the secretory expression of carboxyl-terminal processing protease P9 from A. muciniphila in L. lactis. Our results suggest that genetically engineered L. lactis which expressed P9 may have therapeutic potential for the treatment of diabetes, obesity and other metabolic disorders.

Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Establishing and Monitoring an Effective Management of Benign Nontoxic Multinodular Goitre in Kuwait "Utilizing Two Different Dose Levels of Recombinant Human TSH in Combination with Radioactive Iodine".

INTRODUCTION: rhTSH-assisted radioiodine therapy of multinodular goiter is not fully known and only a few studies, with a limited number of patients have evaluated the effect of rhTSH assisted radioiodine therapy beyond 1 year. Though there is an effective and safe management of benign non-toxic MNG available, it is not applicable to all patient categories in Kuwait covering the impact of the past environmental events (Gulf War) and the genetic relation. The proposed project aims to address those points raised, that is exclusive to the Kuwait population. MATERIALS AND METHODS: In this cohort study, 2 groups of patients, group one (G1) and group two (G2) patients (N=50, ≥18 years old) went undergo evaluated according to a proposed criteria followed by FNA to exclude cancer, toxicity and those who have refused surgery. All patients had a CT scan, TSH, T3, T4 and CBC and complete biomedical tests at a 6-months interval during the treatment period and the follow up. The Volumetric application of GE 670 SPECT/CT (i.e. Xeleris) and in-house developed MATLAB used for quantitative measurement. All patients had a 131-I uptake at baseline and 24 intervals post intramuscular a single dose of 0.3mg or 0.1mg (group 1, group 2) of rhTSH. RESULTS: There was no significant difference in TSH levels at 24-month follow-up between the two groups (p=0.327), whereas there was a statistically significant difference at the baseline and at the 6-months interval between the 2 groups for T4. Post treatment follow up at the 24-hour time point, Group 1 displayed significantly higher uptake than Group 2 (G1:41.74 ± 6.27 vs. G2:34.80 ± 3.84, p < 0.001). The change in I131 uptake from baseline to 24 hours was significantly greater in Group 1compared to Group 2 (p < 0.001). The ROC analysis (AUC) post treatment indicated an excellent discriminatory power for AUC (0.960) and (p < 0.001). There was a much better correlation posttreatment between BMI and thyroid volume for group 1 (R2=0.661) than for group 2 (R2=0.008). Our results suggest 42.1% thyroid volume reduction for group 1 and 20% for group 2. CONCLUSION: The study underscores the potential benefits of the higher rhTSH dose (0.3mg) in managing multinodular non-toxic goiter for the Kuwaiti population and the region considering the impact of dietary, and experience to the drastic environmental exposure.

Protein purification via consecutive histidine-polyphosphate interaction.

While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.

CD5L as a promising biological therapeutic for treating sepsis.

Sepsis results from systemic, dysregulated inflammatory responses to infection, culminating in multiple organ failure. Here, we demonstrate the utility of CD5L for treating experimental sepsis caused by cecal ligation and puncture (CLP). We show that CD5L's important features include its ability to enhance neutrophil recruitment and activation by increasing circulating levels of CXCL1, and to promote neutrophil phagocytosis. CD5L-deficient mice exhibit impaired neutrophil recruitment and compromised bacterial control, rendering them susceptible to attenuated CLP. CD5L-/- peritoneal cells from mice subjected to medium-grade CLP exhibit a heightened pro-inflammatory transcriptional profile, reflecting a loss of control of the immune response to the infection. Intravenous administration of recombinant CD5L (rCD5L) in immunocompetent C57BL/6 wild-type (WT) mice significantly ameliorates measures of disease in the setting of high-grade CLP-induced sepsis. Furthermore, rCD5L lowers endotoxin and damage-associated molecular pattern (DAMP) levels, and protects WT mice from LPS-induced endotoxic shock. These findings warrant the investigation of rCD5L as a possible treatment for sepsis in humans.

Effects of concurrent chemoradiotherapy with or without Endostar on the regression of retropharyngeal lymph node and prognosis of patients with locally advanced nasopharyngeal carcinoma: a retrospective study.

BACKGROUND AND PURPOSE: To investigate the effect of combining Endostar with concurrent chemoradiotherapy (ECCRT) compared to concurrent chemoradiotherapy (CCRT) on the regression rate of retropharyngeal lymph nodes (RLNs) and the relationship between regression rate of RLNs and prognosis of patients with locally advanced nasopharyngeal carcinoma (LANPC). METHODS: A total of 122 LANPC patients with RLNs metastasis were included. Metastatic RLNs were delineated both before and after treatment slice by slice on the magnetic resonance images cross-section. The regression rate of RLNs, adverse effects (AE) were evaluated. The median regression rate of RLNs was taken as the cut-off value, and the patients were furtherly divided into high regression rate (HRR) group and low regression rate (LRR) group, then survival times were evaluated. RESULTS: The median regression rates of RLNs in the ECCRT and CCRT groups were 81% and 50%, respectively (P < 0.001). There was no statistically significant difference in the incidence of grade 3/4 AEs between the two groups, except for oral mucositis (ECCRT 26.23% vs. CCRT 44.26%, P = 0.037). The 3-year overall survival (OS), progression-free survival (PFS), distant metastasis-free survival (DMFS) and locoregional failure-free survival (LRFFS) rates in the HRR and LRR groups were 85.48% and 86.67% (P = 0.983), 80.65% and 68.33% (P = 0.037), 83.87% and 85% (P = 0.704), 93.55% and 81.67% (P = 0.033), respectively. CONCLUSIONS: Patients in the ECCRT group had higher regression rates of RLNs and lower incidence of severe oral mucositis. Furthermore, patients in the HRR group had a better 3-year PFS and LRFFS rate than those in the LRR group.

Effectiveness and safety of interferon α-2a combined with phototherapy for patients with early-stage mycosis fungoides - a single-arm prospective study in 13 patients.

Background: Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma.Objectives: This study was conducted to evaluate efficacy and safety of interferon (IFN) α-2a combined with phototherapy for early-stage MF.Methods: Thirteen patients with early-stage MF received subcutaneous injections of IFN α-2a at 3 million IU combined with phototherapy three times per week for 6 months. Treatment efficacy was measured by changes in body surface area (BSA) score and modified severity-weighted assessment tool (mSWAT) score at 1, 3, and 6 months after treatment. Histopathologic examinations of skin lesions were performed before and after treatment.Results: After 3 months of treatment, all 13 patients achieved a partial response, and BSA and mSWAT scores were significantly lower than those at baseline (p < 0.001). After 6 months, BSA and mSWAT scores were significantly lower than those at baseline (p < 0.001) and after 3 months (p < 0.05). Eleven patients achieved complete remission and two patients achieved a partial response (overall response rate, 100%). Histopathologic examination showed a significant decrease in the number of atypical lymphocytes in both epidermis and dermis. No severe adverse effects occurred.Conclusion: IFN α-2a in combination with phototherapy may be an effective and safe alternative modality for early-stage MF.

Inhibitory effect of recombinant tyrosine­sulfated madanin­1, a thrombin inhibitor, on the behavior of MDA­MB­231 and SKOV3 cells in vitro.

Thrombin, which plays a crucial role in hemostasis, is also implicated in cancer progression. In the present study, the effects of the thrombin­targeting recombinant tyrosine­sulfated madanin­1 on cancer cell behavior and signaling pathways compared with madanin­1 wild­type (WT) were investigated. Recombinant madanin­1 2 sulfation (madanin­1 2S) and madanin­1 WT proteins were generated using Escherichia coli. SKOV3 and MDA­MB­231 cells were treated with purified recombinant proteins with or without thrombin stimulation. Migration and invasion of cells were analyzed by wound healing assay and Transwell assay, respectively. Thrombin markedly increased cell migration and invasion in both SKOV3 and MDA­MB­231 cells, which were significantly suppressed by madanin­1 2S (P<0.05). Madanin­1 2S also significantly suppressed thrombin­induced expression of phosphorylated (p)­Akt and p­extracellular signal­regulated kinase in both cell lines (P<0.05), whereas madanin­1 WT had no effect on the expression levels of these proteins in MDA­MB­231 cells. Furthermore, madanin­1 2S significantly reversed the effects of thrombin on E­cadherin, N­cadherin and vimentin expression in MDA­MB­231 cells (P<0.05), whereas madanin­1 WT did not show any effect. In conclusion, madanin­1 2S suppressed the migration and invasion of cancer cells more effectively than madanin­1 WT. It is hypothesized that inhibiting thrombin via the sulfated form of madanin­1 may be a potential candidate for enhanced cancer therapy; however, further in vivo validation is required.

Expression of soluble moloney murine leukemia virus-reverse transcriptase in Escherichia coli BL21 star (DE3) using autoinduction system.

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96 h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4 U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.

Development of a latex agglutination test based on VH antibody fragment for detection of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is an important porcine pathogen that causes diseases in both swine and human. For rapid SS2 identification, a novel latex agglutination test (LAT) based on heavy-chain variable domain antibody (VH) was developed. Firstly, the soluble 47B3 VH antibody fragment from a phage display library, in which cysteine residues were engineered at the C-terminus, was expressed in Escherichia coli. The purified protein was then gently reduced to form monomeric soluble 47B3 VH subsequently used to coat with latex beads by means of site-specific conjugation. The resulting VH-coated beads gave a good agglutination reaction with SS2. The LAT was able to distinguish S. suis serotype 2 from serotype 1/2, which shares some common sugar residues, and showed no cross-reaction with other serotypes of S. suis or other related bacteria. The detection sensitivity was found to be as high as 1.85x106 cells. The LAT was stable at 4°C for at least six months without loss of activity. To the best of our knowledge, this is the first LAT based on a VH antibody fragment that can be considered as an alternative for conventional antibody-based LAT where VHs are the most favored recombinant antibody.

Updated Evaluation of Agalsidase Alfa Enzyme Replacement Therapy for Patients with Fabry Disease: Insights from Real-World Data.

The clinical use of agalsidase alfa as enzyme replacement therapy (ERT) for Fabry disease (FD) has spread since 2001, and a large body of evidence of its effectiveness has been collected. This review presents the clinical and laboratory results achieved with agalsidase alfa, which has been published in the literature. Agalsidase alfa infusion slows down or stops the progression of renal damage, expressed by reduction or stabilization of the annual decline of the glomerular filtration rate; yearly decrease of glomerular filtration rate (slope) sometimes is reduced until its stabilization. ERT prevents or reduces the occurrence of hypertrophic cardiomyopathy or slows the increase over time if it is already present. Moreover, regarding neurological manifestations, ERT improves neuropathic pain and quality of life, and recent data indicated that it may also prevent the burden of cerebrovascular disease. In addition to ERT's clinical benefits, crucial topics like the most appropriate time to start therapy and the role of anti-drug antibodies (ADA) are analyzed. Treatment with agalsidase alfa in patients with FD substantially improves their outcomes and enhances their quality of life in patients with FD.

Chromatography affinity resin with photosynthetically-sourced protein A ligand.

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.

Efficacy and safety of intracoronary pro-urokinase combined with low-pressure balloon pre-dilatation during percutaneous coronary intervention in patients with anterior ST-segment elevation myocardial infarction.

BACKGROUND: The efficacy and safety of low-pressure balloon pre-dilatation before intracoronary pro-urokinase (pro-UK) in preventing no-reflow during percutaneous coronary intervention (PCI) remains unknown. This study aimed to evaluate the clinical outcomes of intracoronary pro-UK combined with low-pressure balloon pre-dilatation in patients with anterior ST-segment-elevation myocardial infarction (STEMI). METHODS: This was a randomized, single-blind, investigator-initiated trial that included 179 patients diagnosed with acute anterior STEMI. All patients were eligible for PCI and were randomized into two groups: intracoronary pro-UK combined with (ICPpD group, n = 90) or without (ICP group, n = 89) low-pressure balloon pre-dilatation. The main efficacy endpoint was complete epicardial and myocardial reperfusion. The safety endpoints were major adverse cardiovascular events (MACEs), which were analyzed at 12 months follow-up. RESULTS: Patients in the ICPpD group presented significantly higher TIMI myocardial perfusion grade 3 (TMPG3) compared to those in the ICP group (77.78% versus 68.54%, P = 0.013), and STR ≥ 70% after PCI 30 min (34.44% versus 26.97%, P = 0.047) or after PCI 90 min (40.0% versus 31.46%, P = 0.044). MACEs occurred in 23 patients (25.56%) in the ICPpD group and in 32 patients (35.96%) in the ICP group. There was no difference in hemorrhagic complications during hospitalization between the groups. CONCLUSION: Patients with acute anterior STEMI presented more complete epicardial and myocardial reperfusion with adjunctive low-pressure balloon pre-dilatation before intracoronary pro-UK during PCI. TRIAL REGISTRATION: 2019xkj213.

Self-Assembled Recombinant Elastin and Globular Protein Vesicles with Tunable Properties for Diverse Applications.

ConspectusVesicles are self-assembled structures comprised of a membrane-like exterior surrounding a hollow lumen with applications in drug delivery, artificial cells, and micro-bioreactors. Lipid or polymer vesicles are the most common and are made of lipids or polymers, respectively. They are highly useful structures for many applications but it can be challenging to decorate them with proteins or encapsulate proteins in them, owing to the use of organic solvent in their formation and the large size of proteins relative to lipid or polymer molecules. By utilization of recombinant fusion proteins to make vesicles, specific protein domains can be directly incorporated while also imparting tunability and stability. Protein vesicle assembly relies on the design and use of self-assembling amphiphilic proteins. A specific protein vesicle platform made in purely aqueous conditions of a globular, functional protein fused to a glutamate-rich leucine zipper (ZE) and a thermoresponsive elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (ZR) is discussed here. The hydrophobic conformational change of the ELP above its transition temperature drives assembly, and strong ZE/ZR binding enables incorporation of the desired functional protein. Mixing the soluble proteins on ice induces zipper binding, and then warming above the ELP transition temperature (Tt) triggers the transition to and growth of protein-rich coacervates and, finally, reorganization of proteins into vesicles. Vesicle size is tunable based on salt concentration, rate of heating, protein concentration, size of the globular protein, molar ratio of the proteins, and the ELP sequence. Increasing the salt concentration decreases vesicle size by decreasing the Tt, resulting in a shorter coacervation transition stage. Likewise, directly changing the heating rate also changes this time and increasing protein concentration increases coalescence. Increasing globular protein size decreases the size of the vesicle due to steric hindrance. By changing the ELP sequence, which consists of (VPGXG)n, through the guest residue (X) or number of repeats (n), Tt is changed, affecting size. Additionally, the chemical nature of X variation has endowed vesicles with stimuli responsiveness and stability at physiological conditions.Protein vesicles have been used for biocatalysis, biomacromolecular drug delivery, and vaccine applications. Photo-cross-linkable vesicles were used to deliver small molecule cargo to cancer cells in vitro and antigen to immune cells in vivo. pH-responsive vesicles effectively delivered functional protein cargo, including cytochrome C, to the cytosol of cancer cells in vitro, using hydrophobic ion pairing to improve cargo distribution in the vesicles and release. The globular protein used to make the vesicles can be varied to achieve different functions. For example, enzyme vesicles exhibit biocatalysis, and antigen vesicles induce antibody and cellular immune responses after vaccination in mice. Collectively, the development and engineering of the protein vesicle platform has employed amphiphilic self-assembly strategies and rational protein engineering to control physical, chemical, and biological properties for biotechnology and nanomedicine applications.

Exploring a Hirudin variant from nonhematophagous leeches: Unraveling full-length sequence, alternative splicing, function, and potential as a novel anticoagulant polypeptide.

ETHNOPHARMACOLOGICAL RELEVANCE: Leeches exhibit robust anticoagulant activity, making them useful for treating cardiovascular diseases in traditional Chinese medicine. Whitmania pigra, the primary source species of leech-derived medicinal compounds in China, has been demonstrated to possess formidable anticoagulant properties. Hirudin-like peptides, recognized as potent thrombin inhibitors, are prevalent in hematophagous leeches. Considering that W. pigra is a nonhematophagic leech, the following question arises: does a hirudin variant exist in this species? AIM OF THE STUDY: In this study we identified the hirudin-encoding gene (WP_HV1) in the W. pigra genome. The goal of this study was to assess its anticoagulant activity and analyze the related mechanisms. MATERIALS AND METHODS: In this study, a hirudin-encoding gene, WP_HV1, was identified from the W. pigra genome, and its accurate coding sequence (CDS) was validated through cloning from cDNA extracted from fresh W. pigra specimens. The structure of WP_HV1 and the amino acids associated with its anticoagulant activity were determined by sequence and structural analysis and prediction of its binding energy to thrombin. E. coli was used for the expression of WP_HV1 and recombinant proteins with various structures and mutants. The anticoagulant activity of the synthesized recombinant proteins was then confirmed using thrombin time (TT). RESULTS: Validation of the WP_HV1 gene was accomplished, and three alternative splices were discovered. The TT of the blank sample exceeded that of the recombinant WP_HV1 sample by 1.74 times (0.05 mg/ml), indicating positive anticoagulant activity. The anticoagulant activity of WP_HV1 was found to be associated with its C-terminal tyrosine, along with the presence of 9 acidic amino acids on both the left and right sides. A significant reduction in the corresponding TT was observed for the mutated amino acids compared to those of the wild type, with decreases of 4.8, 6.6, and 3.9 s, respectively. In addition, the anticoagulant activity of WP_HV1 was enhanced and prolonged for 2.7 s when the lysine-67 residue was mutated to tryptophan. CONCLUSION: Only one hirudin-encoding variant was identified in W. pigra. The active amino acids associated with anticoagulation in WP_HV1 were resolved and validated, revealing a novel source for screening and developing new anticoagulant drugs.