Rate Of Lysis Research Articles

Biological characteristics and genomic information of a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient and its effects on bacterial biofilm

Biological characteristics and genomic information of a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient and its effects on bacterial biofilm

Potential of Chimeric Antigen Receptor T-Cells in Cancer Therapy.

Novel approaches for targeted delivery like nanoparticles, liposomes, polymer conjugates, etc. with better safety profile needs to be developed for cancer treatment. Chimeric antigen receptors (CAR) with modified thymus cells (T-cells) showed greater potential as a therapy due to its direct effect on immune system responsible for destruction of pathogens and said equivalent to the living drug. On activation of T-cell, it binds to the antigen domains treating refractory or relapsed cancers. The receptors are termed chimeric as it consists of T-cells functioning as well as antigen-binding combined in sole receptor. This therapy showed positive success towards hematological cancers and engineered for specific protein targeting. Though the therapy is associated to several challenges like incompetence towards tumor lysis and cytokine release rate, termination of cytotoxic activity after completion of tumor eradication, etc. The control mechanisms used by CAR T-cells are apoptosis by suicide genes, dual-antigen receptor, ON-switch tumor attack and bispecific molecules as activation switch. In solid tumors, CAR T-cell therapy showed promising signs of efficacy becoming a game-changing cell therapy. CAR T-cells are optimized using different engineering resolutions and lead to broadways for therapy adoption to benefit the cancer patients.

Effect of tannic acid on lysozyme activity through intermolecular noncovalent binding

Abstract Lysozyme (Ly) is a good natural food preservative. However, its enzyme activity is easily influenced by food components, such as tannins. However, the specific mechanism is still unknown. In this study, tannic acid (TA) was used as model tannin to bind with Ly. The interactional properties of Ly-TA complexes were characterized with intrinsic fluorescence intensity, fourier transform infrared spectroscopy, circular dichroism and dynamic light scattering. The enzyme activities of Ly were analyzed by the lysis rate of Micrococcus lysodeikticus. The results indicated that TA can bind well with Ly through hydrophobic, hydrogen and electrostatic interactions, which induced the unfolding of Ly molecule. Consequently, the enzyme activity of Ly was inhibited for conformational changes and masking its active sites by TA, as well as the enzyme stability further declined in pH changes, thermal treatments and high salt concentrations. Our findings shed some light on the possible explanation of negative effects from tannins on the activity of Ly in plant foods.

Blockade of CD47-Sirpα Interactions Improved Myeloid Effector Cell Mediated Lymphoma Cell Killing By the HLA-DR Antibody Apolizumab

Background:Apolizumab (APO) is a humanized IgG1 antibody targeting a HLA-DRβ chain specific epitope, which is expressed by approx. 50 % of B cell lymphomas. The antibody's clinical development was hampered by toxicity, potentially mediated by its strong CDC activity. However, previous studies demonstrated that APO efficiently triggered tumor cell killing by myeloid cells, especially by granulocytes. Myeloid effector cell engagement is strongly regulated by CD47-SIRPα interactions, and blockade of this axis has been demonstrated to improve the efficacy of therapeutic antibodies. Hence, our goal was to characterize the influence of CD47-SIRPα blockade on myeloid cell engagement by APO. Methods: Expression of the APO epitope, CD20 and CD47 on different B lymphoma cell lines was measured by flow cytometry. The capacity of APO and Rituximab (RTX) to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) by mononuclear (MNC) or polymorphonuclear (PMN) effector cells was analysed by 51Cr release assays. Antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages was analysed by fluorescence microscopy. PMN-mediated ADCC and macrophage-mediated ADCP experiments were performed in the presence or absence of the CD47 blocking antibody 5F9-IgG2σ to analyse the impact of CD47-SIRPα blockade on APO-mediated lymphoma cell killing by myeloid cells. Results: As anticipated we observed binding of APO on approx. 50% of the tested B lymphoma cell lines, including Raji (Burkitt's lymphoma), MINO (mantle cell lymphoma) and Carnaval (double hit DLBCL). Focusing on these cell lines, APO's CDC activity was more potent (Raji) or similar (Mino) compared to RTX. Furthermore, APO significantly induced tumor cell lysis by PMN or MNC in 51Cr release assays and achieved higher lysis rates in comparison to RTX. Blocking CD47-SIRPα interactions by 5F9-IgG2σ resulted in significantly increased APO-induced ADCC by PMN against all tested B cell lymphoma lines (Mino: 28.8 ± 10.2 % vs. 41.8 ± 13.3 %; Carnaval: 37.9 ± 17.9 % vs. 55.6 ± 17.6 %; Raji: 23.6 ± 11.7 % vs. 52.4 ± 19.4 % for APO alone vs. APO + 5F9-IgG2σ, respectively, p < 0.05 by ANOVA). However, no ADCC by PMN and RTX was observed - even in the presence of CD47-SIRPα blockade. Moreover, APO mediated similar or even stronger ADCP by macrophages compared to RTX, which could be further enhanced by 5F9-IgG2σ (Figure 1). Conclusions: APO's potent myeloid cell activation could be further enhanced by disruption of CD47-SIRPα interactions. Our observations suggest to generate isotype variants of APO with lower CDC but preserved or enhanced ADCC activity. Subsequently, a combination of APO and CD47-SIRPα blocking strategies may improve B cell lymphoma immunotherapy. Disclosures No relevant conflicts of interest to declare.

Mathematical modelling identifies the role of adaptive immunity as a key controller of respiratory syncytial virus in cotton rats.

Respiratory syncytial virus (RSV) is a common virus that can have varying effects ranging from mild cold-like symptoms to mortality depending on the age and immune status of the individual. We combined mathematical modelling using ordinary differential equations (ODEs) with measurement of RSV infection kinetics in primary well-differentiated human bronchial epithelial cultures in vitro and in immunocompetent and immunosuppressed cotton rats to glean mechanistic details that underlie RSV infection kinetics in the lung. Quantitative analysis of viral titre kinetics in our mathematical model showed that the elimination of infected cells by the adaptive immune response generates unique RSV titre kinetic features including a faster timescale of viral titre clearance than viral production, and a monotonic decrease in the peak RSV titre with decreasing inoculum dose. Parameter estimation in the ODE model using a nonlinear mixed effects approach revealed a very low rate (average single-cell lifetime > 10 days) of cell lysis by RSV before the adaptive immune response is initiated. Our model predicted negligible changes in the RSV titre kinetics at early times post-infection (less than 5 dpi) but a slower decay in RSV titre in immunosuppressed cotton rats compared to that in non-suppressed cotton rats at later times (greater than 5 dpi) in silico. These predictions were in excellent agreement with the experimental results. Our combined approach quantified the importance of the adaptive immune response in suppressing RSV infection in cotton rats, which could be useful in testing RSV vaccine candidates.

Regulatory effect of CLEC2D-CD161 interaction on killing capacity of decidual natural killer cells

Objective To investigate the regulatory effect of CLEC2D-CD161 interaction on killing capacity of decidual natural killer (dNK) cells during early pregnancy and its association with the incidence of recurrent spontaneous abortion (RSA). Methods Decidua tissues were collected from normal pregnancies (n=16) and RSA cases (n=6) at 6-10 gestational weeks in the Department of Obstetrics and Gynecology of Peking University Third Hospital from October 2018 to May 2019. (1) Expressions of CLEC2D and CD161 in decidua from early pregnancy were detected using immunofluorescence. (2) Primary dNK cells were isolated from decidua from early pregnancy. dNK cells pre-treated with CD161 antibody (blocking CD161, B-CD161) were co-cultured with JEG-3 cells which were knocked-down by CLEC2D small interfering RNA (siCLEC2D), followed by killing capacity assessment of dNK cells by cytotoxicity assay and determination of expressions of related molecules by quantitive real-time polymerase chain reaction. (3) Western blot and flow cytometry were used to detect the expression of CLEC2D and CD161 in decidua tissues. Cytotoxicity assay was performed to analyze the killing capacity of dNK cells. T test was used for statistical analysis between normal and RSA cases. Results (1) CLEC2D was mainly expressed in extravillous trophoblast (EVT) cells and CD161 was mainly detected in dNK cells. CD161-positive dNK cells and CLEC2D-positive EVT cells were adjacently located in decidua tissues allowing their interaction. (2) Cytotoxicity assay suggested that CD161 blocking in dNK cells or CLEC2D knockdown in JEG-3 cells could enhance the cytotoxicity of dNK cells. The target cell lysis rates at the effector-target ratios of 40∶1, 20∶1, 10∶1 and 5∶1 in B-CD161 group were (59.12±4.56)%, (25.96±5.44)%, (13.60±8.94)% and (12.53±8.94)%, and in IgG control group were (20.01±1.96)%, (8.51±1.32)%, (3.24±0.75)% and (3.82±1.92)%, respectively. There were significant differences between the two groups at the effector-target ratios of 40∶1 (t=13.922, P 0.05). The target cell lysis rates at the effector-target ratios of 40∶1, 20∶1, 10∶1 and 5∶1 in si-CLEC2D group were (43.37±2.01)%, (32.99±2.08)%, (23.47±1.36)% and (11.48±0.37)%, and in the negative control (NC) group were (15.54±1.46)%, (13.84±1.68)%, (9.94±3.01) and (5.50±0.99)%, respectively. Differences between the two groups at all effector-target ratios were statistically significant (t=19.402, 12.400, 7.093 and 9.842, all P<0.01). Moreover, the expression of dNK killing-related factor granzyme B in the siCLEC2D group was higher than that in the NC group. (3) Compared with the normal pregnancy group, the RSA group showed decreased CD161 expression and increased killing capacity of dNK cells, but no significant difference in CLEC2D expression. Conclusions At early pregnancy, CLEC2D on EVT cells can interact with CD161 on dNK cells, which inhibits the cytotoxicity of dNK cells and induces immune tolerance at the fetal-maternal interface. Decreased expression of CD161 in decidua results in increased cytotoxicity of dNK cells, which may be one of the causes of immune rejection in RSA. Key words: Abortion, habitual; Decidua; Killer cells, natural; Receptors, cell surface; Lectins, C-type; NK cell lectin-like receptor subfamily B

Plasma-Alginate Composite Material Provides Improved Mechanical Support for Stem Cell Growth and Delivery.

Blood plasma-based products have been recently utilized in different tissue engineering applications, ranging from soft tissue repair to bone regeneration. Plasma contains fibrinogen which can be converted to an insoluble fibrin-laden gel in the presence of activated thrombin. In tissue engineering, these plasma-based materials can serve either as a three-dimensional scaffold to deliver therapeutic cells in vivo or as a growth factor-rich supply for tissue regeneration. Unfortunately, plasma-based materials are often mechanically weak and easily deformed, thus limiting their usability in harsh clinical settings. Simpler methods to create sturdier plasma-based materials are therefore needed. To this end, we hypothesized that combining alginate with plasma can create a composite plasma material with improved mechanical properties. Incorporating alginate into plasma produced composite gels with increasing bulk stiffness, as measured by rheology. Specifically, the plasma-alginate composite (PAC) gels with an alginate concentration of 2.86 mg/mL were 10-fold stiffer than pure plasma gels (11 vs 112 Pa). Interestingly, gel lysis rates were unchanged despite increasing alginate concentration (lysis time approximately 50 min). Adipose-derived stem cells cultured in the stiffer PAC gels expressed stemness markers (THY1, ENG, NT5E) at levels comparable to those in the pure plasma gels. Similarly, proangiogenic factor secretion was also constant across all gel conditions. In sum, we envision this PAC gel system will extend the use of plasma gel-based therapies into more rigorous clinical applications.

Modular Assembly of Unique Chimeric Lytic Enzymes on a Protein Scaffold Possessing Anti-Staphylococcal Activity.

Lytic enzymes have been considered as potential alternatives to antibiotics. These enzymes, particularly those that target Gram-positive bacteria, consist of modular cell wall-binding and catalytic domains, which can be shuffled with those of other lytic enzymes to produce unnatural chimeric enzymes. In this work, we report the in vitro shuffling of two different modular domains using a protein self-assembly methodology. Catalytic domains (CD) and cell wall-binding domains (BD) from the bacteriocin lysostaphin (Lst) and a putative autolysin from Staphylococcus aureus (SA1), respectively, were genetically site-specifically biotinylated and assembled with streptavidin to generate 23 permuted chimeras. The specific assembly of a CD (3 equiv) and a BD (1 equiv) from Lst and SA1, respectively [CDL-BDS (3:1)], on a streptavidin scaffold yielded high lytic activity against S. aureus (at least 5.6 log reduction), which was higher than that obtained with either native Lst or SA1 alone. Moreover, at 37 °C, the initial rate of cell lysis was over 3-fold higher than that with free Lst, thereby revealing the unique catalytic properties of the chimeric proteins. In vitro self-assembly of functional domains from modular lytic enzymes on a protein scaffold likely expands the repertoire of bactericidal enzymes with improved activities.

Feasibility of low-dose infusion of alteplase for unsuccessful thrombolysis with urokinase in deep venous thrombosis.

The aim of the present study was to investigate whether the rescue of thrombolysis with the recombinant tissue plasminogen activator (rt-PA) alteplase was an effective and safe therapeutic option in patients who did not respond to urokinase. Between February 2016 and February 2017, 26 patients with deep venous thrombosis (DVT) underwent rescue thrombolysis with alteplase. Unsuccessful thrombolysis with urokinase was defined as a lack of improvement in the degree of thrombotic removal with a lysis rate <50% under one of the following three conditions: Two consecutive venography procedures, administration of >3 million units of total urokinase, or >7 days infusion duration. The thrombus score, lysis rate and post-thrombolysis safety of alteplase, following unsuccessful urokinase thrombolysis were all evaluated. At the end of the unsuccessful urokinase thrombolytic therapy, the mean duration of the perfusion was 6.09±1.60 days, and the mean total dose was (362.5±90.0) ×104 units. No significant difference was detected in the total thrombus score before (7.85±2.40) and at the completion (6.19±2.33) of urokinase thrombolysis (P>0.05). The mean duration of perfusion was 3.36±1.69 days, and the mean total infusion dose was 44.8±22.6 mg for the rescue thrombolysis with alteplase. The mean thrombus score decreased to 1.19±2.10 at the completion of rescue thrombolysis. The alteplase post-thrombolysis scores were significantly decreased compared with those of urokinase thrombolysis (P<0.05). There were 23 (88.5%) patients who achieved a successful lysis rate (grade II/III) following rescue thrombolysis with alteplase, and symptoms of swelling and pain in the affected limbs were significantly improved. Successful thrombolysis rates in patients in the acute (<14 days) and subacute (14-28 days) phases were high (93.3 and 81.8%, respectively; P>0.05). No symptomatic pulmonary embolism or major bleeding occurred during rescue thrombolysis, but minor bleeding complications occurred in 4 cases (15.4%). In conclusion, rescue thrombolysis with alteplase led to an effective and safe outcome in patients with DVT who did not respond to initial thrombolysis with urokinase, and may be a valid and easy alternative treatment option.

Viral Infections Boost Prokaryotic Biomass Production and Organic C Cycling in Hadal Trench Sediments.

Hadal trenches are among the most remote and least explored ecosystems on Earth and can support high benthic microbial standing stocks and activities. However, information on the role of viruses in such ecosystems and their interactions with prokaryotic hosts is very limited. Here, we investigated activities of benthic viruses and prokaryotes and their interactions in three hadal trenches (Japan, Izu-Ogasawara and Mariana trenches) and in their nearby abyssal sites. Our findings reveal that these hadal trenches, compared with the surrounding abyssal sites, support higher abundances and biomasses of prokaryotes. In addition, the high prokaryotic biomasses of hadal trenches could favor high rates of viral infection and cell lysis, especially in the Japan Trench. Hadal viruses can release large amounts of highly labile and promptly available organic material by inducing cell lysis, which could contribute to sustain benthic prokaryotes and decrease their dependency on the enzymatic digestion of the more refractory fraction of sediment organic matter. Our results suggest that this process can contribute to explain the discrepancy between high prokaryote biomass and apparent low efficiency in the utilization of the sedimentary organic matter in the hadal ecosystems. Concluding, hadal trenches may be characterized by a highly dynamic viral component, which can boost prokaryotic biomass production, thereby profoundly influencing the functioning of these remote and extreme ecosystems.

Low immunogenic endothelial cells endothelialize the Left Ventricular Assist Device

Low haemocompatibility of left ventricular assist devices (LVAD) surfaces necessitates anticoagulative therapy. Endothelial cell (EC) seeding can support haemocompatibility, however, the availability of autologous ECs is limited. In contrast, allogeneic ECs are readily available in sufficient quantity, but HLA disparities induce harmful immune responses causing EC loss. In this study, we investigated the feasibility of using allogeneic low immunogenic ECs to endothelialize LVAD sintered inflow cannulas (SIC). To reduce the immunogenicity of ECs, we applied an inducible lentiviral vector to deliver short-hairpins RNA to silence HLA class I expression. HLA class I expression on ECs was conditionally silenced by up to 70%. Sufficient and comparable endothelialization rates were achieved with HLA-expressing or HLA-silenced ECs. Cell proliferation was not impaired by cell-to-Sintered Inflow Cannulas (SIC) contact or by silencing HLA expression. The levels of endothelial phenotypic and thrombogenic markers or cytokine secretion profiles remained unaffected. HLA-silenced ECs-coated SIC exhibited reduced thrombogenicity. In contrast to native ECs, HLA-silenced ECs showed lower cell lysis rates when exposed to allogeneic T cells or specific anti-HLA antibodies. Allogeneic HLA-silenced ECs could potentially become a valuable source for LVAD endothelialization to reduce immunogenicity and correspondingly the need for anticoagulative therapy which can entail severe side effects.

Sonothrombolysis with magnetic microbubbles under a rotational magnetic field

Thrombosis is an extremely critical clinical condition where a clot forms inside a blood vessel which blocks the blood flow through the cardiovascular system. Previous sonothrombolysis methods using ultrasound and microbubbles (MBs) often have a relatively low lysis rate due to the low microbubbles concentration at clot region caused by blood flow in the vessel. To solve this problem, the magnetic microbubbles (MMBs) that can be retained by an outer magnetic field against blood flow are used in this study. Here we report the development of a new method using the rotational magnetic field to trap and vibrate magnetic microbubbles at target clot region and then using an intravascular forward-looking ultrasound transducer to activate them acoustically. In this study, we investigated the influence of different blood flow conditions, vessel occlusion conditions (partial and fully occluded), clot ages (fresh, retracted), ultrasound parameters (input voltage, duty cycle) and rotational magnetic field parameters (amplitude, frequency) on the thrombolysis rate. The results showed that the additional use of magnetic microbubbles significantly enhances in vitro lysis of blood clot.

PB2432 POSSIBLE GENETIC RISK FACTORS OF PULMONARY EMBOLISM IN YOUNG PATIENTS FROM THE NORTH‐WESTERN REGION OF RUSSIA

Background:Pulmonary embolism (PE) and deep‐vein thrombosis (DVT) are the most common clinical manifestations of venous thromboembolism (VTE) which associated with high rates of morbidity and mortality in population. To date, little is known about the factors which could predict PE development in VTE patients. Several studies have shown that fibrin clot structure as well as the rate of clot lysis could influence the risk of PE development. Moreover, nucleotide variations in genes coding for components involved in processes of thrombus formation or its lysis are well‐described. Nevertheless, association between patient's genotype and features of clinical VTE manifestation, in particular, risk of PE development, remains debatable.Aims:To reveal possible association between the alpha‐fibrinogen (FI‐A), factor XIII A‐subunit (FXIII‐A), tissue plasminogen activator (TPA) and plasminogen activator inhibitor type I (PAI‐1) gene polymorphisms and the features of clinical VTE manifestation in patients with early‐onset disease from the North‐Western region of Russia.Methods:We analyzed 243 young patients under 45 years old (119 men and 124 women, mean age – 37.4 years) with at least one confirmed episode of acute VTE. Isolated DVT (“DVT only”) was diagnosed in 154 (63.4%), DVT complicated by PE (“DVT + PE”) – in 53 (21.8%), and isolated PE (“PE only”) – in 36 (14.8%) patients. Genotyping for the FI‐A Thr312Ala, FXIII‐A Val34Leu, PAI‐1 ‐675 4G/5G and TPA 311 bp Ins/Del variations was performed by PCR‐RFLP. To assess intergroup differences in genotype frequencies, the odds ratios (OR), their 95% confidence intervals (CI) and p‐values were calculated by Fisher's exact method.Results:Distributions of the FI‐A, FXIII‐A, PAI‐1 and TPA genotypes in patients with different clinical manifestations of VTE have shown several differences. Frequency of the TPA Del/Del genotype was significantly increased in VTE patients with the signs of PE when compared to those with isolated DVT (31.5% vs. 18.8%, respectively, OR = 2.0; 95% CI: 1.1‐3.6; p = 0.029). Homozygosity for the TPA Del allele was most prevalent in patients with isolated PE (38.9% vs. 18.8% in the group “DVT only”, OR = 2.7; 95% CI: 1.3‐6.0; p = 0.014). In this group, the frequency of the FI‐A 312 Ala/Ala variant was 2‐fold increased when compared to patients with isolated DVT (22.2% vs. 11.0%, respectively, OR = 2.3; 95% CI: 0.9‐5.9; p = 0.098). In patients with the signs of PE, homozygosity for the FXIII‐A 34Leu variant was observed more frequently than in the “DVT only” group (11.2% vs. 6.5%, respectively, OR = 1.8; 95% CI: 0.7‐4.6; p = 0.23). At the same time, the frequency of heterozygous FXIII‐A genotype was significantly increased in patients with isolated DVT when compared to “DVT + PE” group (48.7% vs. 30.2%, respectively, OR = 2.2; 95% CI: 1.1‐4.3; p = 0.024).Summary/Conclusion:TPA Del/Del genotype is associated with the risk of PE development in young VTE patients from the North‐Western region of Russia, whereas heterozygous FXIII‐A variant (34 Val/Leu) could protect from PE in patients with DVT. Role of the FI‐A 312 Ala/Ala and FXIII‐A 34 Leu/Leu variants as possible determinants of different clinical course of VTE needs further clarification.

Carbon monoxide releasing molecule enhances coagulation and decreases fibrinolysis in canine plasma exposed to Crotalus viridis venom in vitro and in vivo.

Carbon monoxide releasing molecule-2 (CORM-2), an emerging therapeutic in human medicine, enhances plasmatic coagulation and attenuates fibrinolysis in vitro in human, rabbit and horse plasma and ameliorates hypocoagulation and hyperfibrinolysis secondary to venom exposure in human plasma in vitro. Fibrinogenases in rattlesnake venom cause decreased clot strength, and in the presence of tissue plasminogen activator (tPA) in vitro, a markedly increased rate of clot lysis. CO interacts with a haem group on fibrinogen, changing its configuration so that the fibrin clot is strengthened and more resistant to fibrinolysis. We hypothesized that CORM-2 enhances coagulation and attenuates fibrinolysis in canine plasma exposed to Cviridis venom. We measured the effects of Cviridis venom on clot strength, rates of coagulation and fibrinolysis in both pooled canine plasma and plasma from individual naturally envenomed dogs, with and without CORM-2, using thromboelastography (TEG). We tested venom effects on coagulation using tissue factor (TF) activated TEG and on both coagulation and fibrinolysis using TF-activated TEG with added tPA. We found that 17.9µg/mL of venom causes a mean 26.4% decrease in clot strength, a 61.8% decrease in maximum rate of thrombus generation, 75% faster clot lysis, a 226% increase in maximum rate of lysis and a 92% decrease in total clot life span (CLS). CORM-2 ameliorated these effects, increasing CLS in the presence of venom by 603%. Additionally, we showed that CORM-2 has similar effects in vitro on plasma from naturally envenomed dogs, showing promise as an adjunct therapy for snake envenomation.

Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants.

Frequently measured mammalian cell culture process indicators include viability and total cell concentration (TCC). Cell lysis, an additional important process characteristic that substantially contributes to the overall product purity profiles, is often not addressed in detail. In the present study, an inexpensive and simple application of the Bradford assay is developed to determine the residual protein content (RPC) in cell culture supernatants. The reliability and reproducibility of the method are tested in a long-term study and compared with lysis quantification via the DNA measurement. The results show that its performance is more robust and accurate over time and the respective concentration range. Additionally, both methods are used for cell lysis process monitoring in a recombinant Chinese hamster ovary fed-batch process. In the presented process, by applying the established assay, the lysis rate k DL is determined to be constant over time at 4.6 × 10 -4 lysed cell concentration (LCC) per TCC and time (LCC/TCC/h). In contrast, DNA data did not confirm the constant lysis rate due to variations of the content per cell during cultivation. Thus, information on the RPC can facilitate the determination of the optimal harvest time point with respect to purity and in improving process characterization.

Time-Resolved Single-Cell Lysis Kinetics of Metal Nanoparticle-Infused Biopolymer Matrices

Bacterial infections continue to be a growing concern in healthcare facilities due to the increasing number of multi-drug resistant bacteria. The extensive use of antibiotics has weakened the potency and effectiveness of these drugs, consequently promoting bacterial infections and extending healing time. Therefore, there is an immediate demand to find an alternate method to combat the spread of bacterial infections. Metal nanoparticles (MNP) have proven to be an effective antibacterial agent against antibiotic-resistant bacteria, and when incorporated into polymeric substrates, have exhibited high toxicity towards bacterial cells. Current research proposes that MNP interact with the bacterial cell wall, undergo redox processes to form ions, and the ions diffuse into the cell resulting in disruption of membrane-bound proteins. This results in the inhibition of cell replication or cell lysis. However, existing methods of antimicrobial susceptibility testing, such as broth dilution assays and disk diffusion, fail to provide a greater understanding of the time scale required for the single cell lysis process to occur. More specifically, the conventional methods have substrate and agent limitations, as well as assume homogeneity in the bacterial population, therefore making the techniques an ineffective method for probing the cell lysis mechanism of MNP-infused biopolymer matrices. To further investigate the mechanistic action of metal nanocomposites, this research utilizes epi-fluorescence optical tweezers for antibacterial characterization. More specifically, a single E. coli cell was optically trapped, non-covalently docked to MNP-infused alginate matrix, and the cell viability kinetics were monitored in situ. Initial results showed that antibacterial activity of Cu and Ag functionalized nanocomposites was related to the metal concentration and size of colloidal NP. Ag incorporated polymers exhibited greater antibacterial effectiveness when compared to Cu potentially due to the role of Cu in cellular respiration processes. Results further indicate that the size of the MNP does impact the rate of single cell lysis, which may be related to the redox kinetics at the cell/MNP interface.

Rhamnolipid induced deagglomeration of anaerobic granular biosolids for energetically feasible ultrasonic homogenization and profitable biohydrogen

The present study exposes the effect of deagglomeration using rhamnolipid on anaerobic granular biosolids (AGB) followed by ultrasonic homogenization for effective biohydrogen production. Rhamnolipid was used to remove the extracellular polymeric substance bound over the surface of AGB to increase the rate of biogranular lysis during ultrasonic homogenization. Extracellular polymeric substance (EPS) removal was achieved at an optimum rhamnolipid dosage of 0.04 g Rh/g SS. Ultrasonic homogenization (UH) of AGB demands 27016 kJ/kg TS of specific energy to achieve 16.8% and 13.9% of biogranular lysis and biosolids reduction, respectively. However, rhamnolipid-alkaline pH induced ultrasonic homogenization (RAUH) demand lesser (12607 kJ/kg TS) and achieves greater biogranular lysis (25.4%) and biosolids reduction (20.7%). RAUH significantly saves the net energy. Exponential first order kinetic analysis was done to evaluate and compare the biohydrogen production potential of RAUH with that of UH. The biohydrogen production was found to be 55.1 mL H2/g COD and 36.7 mL H2/g COD for RAUH and UH respectively. A higher positive net energy of 2.62 kWh/kg AGB was achieved by RAUH when compared to UH (−3.49 kWh/kg anaerobic granular biosolids).

Seasonal Variability and Drivers of Microzooplankton Grazing and Phytoplankton Growth in a Subtropical Estuary

Rates of microzooplankton grazing and phytoplankton growth are seldom measured with respect to time, yet such estimates may better reflect temporal variability in coastal phytoplankton communities and offer insight into mechanisms that control populations. To assess seasonal patterns in rates, we performed 41, weekly dilution experiments over a full year in the Skidaway River Estuary (GA), measuring rates of phytoplankton growth, microzooplankton grazing, and viral lysis based on total chlorophyll and group-specific abundances (Synechococcus spp., picoeukaryotes, and nanoeukaryotes). Seasonal variability in microzooplankton grazing (0-2.11 d-1) and phytoplankton growth rates (0-2.43 d-1) was observed, with highest values typically recorded in summer and lowest in winter. Grazing pressure was strongest in winter-spring, as phytoplankton accumulation rates were often negative (-0.16-0.28 d-1). Rates varied similarly over seasons for chlorophyll, pico-, and nanoeukaryotes, while rates on Synechococcus spp. were rarely significant in dilutions and did not follow seasonal trends. Few experiments (7%) yielded significant rates of viral lysis. Temperature correlated most strongly with phytoplankton growth rates (R2 = 0.46-0.56), but not grazing rates (R2 = 0.11-0.27), which were more likely driven by observed seasonal shifts in plankton community composition (e.g. fall diatom blooms). Establishing temporal rate measurements is critical to identify factors that drive phytoplankton growth and mortality and accurately predict shifts in phytoplankton population dynamics and food web processes within marine systems.

Generation of porcine Pasteurella multocida ghost vaccine and examination of its immunogenicity against virulent challenge in mice

Pasteurella multocida (PM) causes a varity of clinical manifestation in domestic animals, even acute death. Vaccination is among effective strategy to prevent and control PM-related diseases. Bacterial ghosts (BGs) are empty bacterial envelopes, which sustain subtle antigenic comformation in bacterial outer-membrane and exhibit higher efficacy compared to inactivated vaccines. Here, a BG vaccine generated from the porcine PM reference strain CVCC446 (serotype B:2) was prepared upon lysis by E protein of bacteriophage PhiX174, and the safety and immunogenicity were evaluated its in a mouse model. Lysis rate was in 99.99% and the BG vaccine was completely inactivated by addition of freeze-dry procedure. Mice were immunized subcutaneously twice in 2-week intervals with BGs, or BGs plus adjuvant, or formalin-inactivated PM or an adjuvant control. Mice inoculated twice with BGs vaccines generated higher titer of antibodies, interleukin 4 and gamma interferon than those in the inactivated vaccine group or adjuvant placebo group (P < 0.05). CD4+ and CD8+ T lymphocyte levels in spleen were higher in both BG groups than inactivated vaccine group or adjuvant group. Mice administered with the BGs plus adjuvant were completely protected against intraperitoneal challenge with 10 × LD50 dose of virulent isolate and exhibited decreased tissue lesion and lower bacterial loads, which was superior to the inactivated vaccine. The results demonstrated safety of the BG vaccine and primary immunogenicity in a mouse model, suggesting a potential of further evaluation in a pig model and vaccine candidate.

Physico-Chemical and Biological Properties of Biosimilar and Reference Tissue Plasminogen Activator Products

Recombinant tissue plasminogen activator (international nonproprietary name — alteplase) which was developed by «GENERIUM» (Russia) and received a marketing authorisation in Russia is completely analogous to Actilyse® which is used to treat medical conditions accompanied by thrombosis, such as acute myocardial infarction, pulmonary embolism, and ischemic stroke. The aim of the study was to carry out a comprehensive comparison of physico-chemical and biological properties of Revelyse® and the reference product Actilyse® in order to assess their biosimilarity. Materials and Methods: comparative peptide mapping and determination of comparability of chromatographic profiles of tryptic hydrolysates was performed using RP-HPLC and massspectrometry; the molecular weight distribution was determined by mass-spectrometry and polyacrylamide gel electrophoresis (Laemmli method). The purity and homogeneity of products as well as the content of related impurities (oligomers and fragments) were determined using gel filtration; N-glycosylation profile was analysed by hydrophilic HPLC, total sialic acid was quantified by the Svennerholm resorcinol method. Protein binding to fibrin and human fibrinogen was assessed by surface plasmon resonance, and the specific activity was compared by fibrin clot lysis. Results: the research demonstrated a complete overlap of the products’ peptide maps, which indicates the identity of аlteplase amino acid sequences in the two medicines being compared. The authors of the study also determined the molecular weight and the content of the intact single-stranded form of the protein, and quantified post-translational modifications, the content of sialic acids and neutral sugars. The analysis of the N-glycosylation profile revealed insignificant differences in the percentage of multiantenna complex glycans. The specificity of alteplase was evaluated by analysing the formation of protein complexes with natural alteplase ligands – fibrin and plasminogen activator inhibitor-1, but no significant differences were found. The comparison of specific activation of plasminogen fibrinolytic activity was performed based on the results of the assay analysing the fibrin clot lysis rate, and it demonstrated comparability of Revelyse® and Actilyse®. Conclusions: comparative experimental studies have shown no differences in the structure, charge distribution heterogeneity, impurities content, and specific activity of alteplase as a component of Revelyse® and the reference product Actilyse®, which leads to the conclusion that they are similar in terms of physicochemical and biological properties.