Rapid Cell Lysis Research Articles

Engineering EPS in Halomonas bluephagenesis leads to self-flocculation and filamentation for convenient downstream processing

The halophile Halomonas bluephagenesis has established itself as a cost-effective chassis in industrial bioproduction. However, downstream processing (DSP) expense is a persistent challenge. This study uses genetic knockout of exopolysaccharides (EPS-KO) in six industrially relevant H. bluephagenesis strains, each producing distinct polyhydroxyalkanoate (PHA), to evaluate their impact on reducing DSP costs. Remarkably, EPS-KO cells exhibited an unprecedented filamentous morphology, reaching lengths over 50 times (10–75 μm) that of standard cells. This unexpected behavior initially posed a challenge to industrial applicability due to rapid cell lysis under fermentation agitation. Systematic media supplementation of salt, carbon source, nitrogen, and essential minerals significantly influenced morphology, and tailored nutrient schemes that mitigated filamentation while maintaining production levels were designed. Scaled production in a 5000 L bioreactor demonstrated 85 % PHA and 96 g/L cell dry weight after 40 h fermentation, consistent with non-KO cells. During fermentation, EPS-KO enhanced cell-wall penetrability, reducing the need for high-cost γ-butyrolactone by 40 %. Additionally, a new innovative 5000 L FlipFlow bioreactor design cut rotation speed by 50 % and air input by 25 %. During DSP, centrifugation time decreased tenfold due to rapid cell self-flocculation (15–30 min), and the water and enzyme required for PHA purification was reduced by 30 % and 35 %, respectively. This study highlights the use of EPS-KO cells, a controlled media strategy, and low shear force FlipFlow bioreactor to successfully reduce DSP costs without sacrificing productivity. These novel findings represent a notable advancement in cost-saving strategies while presenting the intriguing discovery of media-controlled filamentous cell behavior.

Inhibition of multiple staphylococcal growth states by a small molecule that disrupts membrane fluidity and voltage.

New molecular approaches to disrupting bacterial infections are needed. The bacterial cell membrane is an essential structure with diverse potential lipid and protein targets for antimicrobials. While rapid lysis of the bacterial cell membrane kills bacteria, lytic compounds are generally toxic to whole animals. In contrast, compounds that subtly damage the bacterial cell membrane could disable a microbe, facilitating pathogen clearance by the immune system with limited compound toxicity. A previously described small molecule, D66, terminates Salmonella enterica serotype Typhimurium (S. Typhimurium) infection of macrophages and reduces tissue colonization in mice. The compound dissipates bacterial inner membrane voltage without rapid cell lysis under broth conditions that permeabilize the outer membrane or disable efflux pumps. In standard media, the cell envelope protects Gram-negative bacteria from D66. We evaluated the activity of D66 in Gram-positive bacteria because their distinct envelope structure, specifically the absence of an outer membrane, could facilitate mechanism of action studies. We observed that D66 inhibited Gram-positive bacterial cell growth, rapidly increased Staphylococcus aureus membrane fluidity, and disrupted membrane voltage while barrier function remained intact. The compound also prevented planktonic staphylococcus from forming biofilms and a disturbed three-dimensional structure in 1-day-old biofilms. D66 furthermore reduced the survival of staphylococcal persister cells and of intracellular S. aureus. These data indicate that staphylococcal cells in multiple growth states germane to infection are susceptible to changes in lipid packing and membrane conductivity. Thus, agents that subtly damage bacterial cell membranes could have utility in preventing or treating disease.IMPORTANCEAn underutilized potential antibacterial target is the cell membrane, which supports or associates with approximately half of bacterial proteins and has a phospholipid makeup distinct from mammalian cell membranes. Previously, an experimental small molecule, D66, was shown to subtly damage Gram-negative bacterial cell membranes and to disrupt infection of mammalian cells. Here, we show that D66 increases the fluidity of Gram-positive bacterial cell membranes, dissipates membrane voltage, and inhibits the human pathogen Staphylococcus aureus in several infection-relevant growth states. Thus, compounds that cause membrane damage without lysing cells could be useful for mitigating infections caused by S. aureus.

Field and laboratory studies of fluorescence-based technologies for real-time tracking of cyanobacterial cell lysis and potential microcystins release

Elevated levels of dissolved microcystins (MCs) in source water due to rapid cell lysis of harmful cyanobacterial blooms may pose serious challenges for drinking water treatment. Catastrophic cell lysis can result from outbreaks of naturally-occurring cyanophages – as documented in Lake Erie during the Toledo water crisis of 2014 and in 2019, or through the application of algaecides or water treatment chemicals. Real-time detection of cyanobacterial cell lysis in source water would provide a valuable tool for drinking water plant and reservoir managers. In this study we explored two real-time fluorescence-based devices, PhycoSens and PhycoLA, that can detect unbound phycocyanin (uPC) as a potential indication of cell lysis and MCs release. The PhycoSens was deployed at the Low Service pump station of the City of Toledo Lake Erie drinking water treatment plant from July 15 to October 19, 2022 during the annual cyanobacteria bloom season. It measured major algal groups and uPC in incoming lake water at 15-min intervals during cyanobacteria dominant and senescence periods. Intermittent uPC detections from the PhycoSens over a three-month period coincided with periods of increasing proportions of extracellular MCs relative to total (intracellular and extracellular) MCs, indicating potential for uPC use as an indicator of cyanobacterial cell integrity. Following exposures of laboratory-cultured MCs-producing Microcystis aeruginosa NIES-298 (120 μg chlorophyll/L) to cyanophage Ma-LMM01, copper sulfate (0.5 and 1 mg Cu/L), sodium carbonate peroxyhydrate (PAK® 27, 6.7 and 10 mg H2O2/L), and potassium permanganate (2.5 and 4 mg/L), appearance of uPC coincided with elevated fractions of extracellular MCs. The PhycoLA was used to monitor batch samples collected daily from Lake Erie water exposed to algaecides in the laboratory. Concurrence of uPC signal and surge of dissolved MCs was observed following 24-h exposures to copper sulfate and PAK 27. Overall results indicate the appearance of uPC is a useful indicator of the onset of cyanobacterial cell lysis and the release of MCs when MCs are present.

The effect of viral infection on the Black Sea microalgae Tetraselmis viridis: the role of nutrients and copper ions.

The TvV-SM2 virus, isolated from the coastal waters of the Black Sea, causes lysis of its host, the algae Tetraselmis viridis (Chlorophyta). Under optimal conditions for nutrients, an increase in the initial abundance of algae cells by four times caused a 3-fold reduction in the latent period of viral infection. During the period of the most rapid cell lysis of T. viridis , nitrogen deficiency leads to a decrease in the average daily rate of death of cells affected by the virus by 3.2times relative to the replete conditions, while in the case of phosphorus deficiency, this process slows down by up to 2.4times. Under deplete conditions, the rate of cell death was only 34% lower than under replete conditions. The effect of copper ions (100μgL-1 ) on the viral suspension for 6h led to the complete suppression of its activity. In the presence of the host of this virus, its activity is only partially suppressed. As a result, cell lysis under the influence of a viral infection occurred in two stages. The first stage was noted only during the first 6h of the experiment. The second main stage took place within 78-170h. This study showed that in conditions of nutrient deficiency and in the presence of copper ions in seawater, the impact of viruses on microalgae will be weaker.

Multi-functional single cell manipulation method based on a multichannel micropipette

Cell manipulation techniques that provide versatility in handling various cell types and performing diverse manipulations are essential for advancing biological research. This study presents a novel and adaptable approach utilizing a multifunctional micropipette for precise single-cell operations, including three-dimensional (3D) manipulation, trapping, lysis, and aspiration. The multifunctional micropipette comprises two liquid metal-based channels with high electrical conductivity, along with a negative pressure-based aspiration channel. By utilizing the liquid metal electrodes, the system enables the application of electrical stimulation, inducing both dielectrophoretic force and electroporation on cells. Through the implementation of dielectrophoretic force and integration with a 3D-movable stage, single cells can be patterned, manipulated in 3D, accurately trapped, and separated with a minimal adjacent distance of 10 µm. By utilizing the electroporation phenomenon, rapid lysis of suspension cells can be achieved within 50 ms, while adherent cells can be lysed within 200 ms, utilizing a square-wave electrical signal with a frequency of 1 kHz and an amplitude of 40 Vpp. Furthermore, the study explores the impact of electrode size on the lysis of adherent cell groups. Additionally, the utilization of negative pressure for lysate aspiration is also demonstrated. In summary, the reported technique is versatile for single cell manipulation.

Systemic application of bone-targeting peptidoglycan hydrolases as a novel treatment approach for staphylococcal bone infection.

The rising prevalence of antimicrobial resistance in S. aureus has rendered treatment of staphylococcal infections increasingly difficult, making the discovery of alternative treatment options a high priority. Peptidoglycan hydrolases, a diverse group of bacteriolytic enzymes, show high promise as such alternatives due to their rapid and specific lysis of bacterial cells, independent of antibiotic resistance profiles. However, using these enzymes for the systemic treatment of local infections, such as osteomyelitis foci, needs improvement, as the therapeutic distributes throughout the whole host, resulting in low concentrations at the actual infection site. In addition, the occurrence of intracellularly persisting bacteria can lead to relapsing infections. Here, we describe an approach using tissue-targeting to increase the local concentration of therapeutic enzymes in the infected bone. The enzymes were modified with a short targeting moiety that mediated accumulation of the therapeutic in osteoblasts and additionally enables targeting of intracellularly surviving bacteria.

Rapid acoustofluidic mixing by ultrasonic surface acoustic wave-induced acoustic streaming flow

Ultrasonic surface acoustic wave (SAW)-induced acoustic streaming flow (ASF) has been utilized for microfluidic flow control, patterning, and mixing. Most previous research employed cross-type SAW acousto-microfluidic mixers, in which the SAWs propagated perpendicular to the flow direction. In this configuration, the flow mixing was induced predominantly by the horizontal component of the acoustic force, which was usually much smaller than the vertical component, leading to energy inefficiency and limited controllability. Here, we propose a vertical-type ultrasonic SAW acousto-microfluidic mixer to achieve rapid flow mixing with improved efficiency and controllability. We conducted in-depth numerical and experimental investigations of the vertical-type SAW-induced ASF to elucidate the acousto-hydrodynamic phenomenon under varying conditions of total flow rate, acoustic wave amplitude, and fluid viscosity conditions. We conducted computational fluid dynamics simulations for numerical flow visualization and utilized micro-prism-embedded microchannels for experimental flow visualization for the vertical SAW-induced ASF. We found that the SAW-induced vortices served as a hydrodynamic barrier for the co-flow streams for controlled flow mixing in the proposed device. For proof-of-concept application, we performed chemical additive-free rapid red blood cell lysis and achieved rapid cell lysis with high lysis efficiency based on the physical interactions of the suspended cells with the SAW-induced acoustic vortical flows. We believe that the proposed vertical-type ultrasonic SAW-based mixer can be broadly utilized for various microfluidic applications that require rapid, controlled flow mixing.

Intracellular replication of Pseudomonas aeruginosa in epithelial cells requires suppression of the caspase-4 inflammasome.

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

Oncolytic adenovirus coding for bispecific T cell engager against human MUC-1 potentiates T cell response against solid tumors

Immunotherapy with bispecific Tcell engagers has shown efficacy in patients with hematologic malignancies and uveal melanoma. Antitumor effects of bispecific Tcell engagers in most solid tumors are limited due to their short serum half-life and insufficient tumor concentration. We designed a novel serotype 5/3 oncolytic adenovirus encoding a human mucin1 antibody and the human CD3 receptor, Ad5/3-E2F-d24-aMUC1aCD3 (TILT-321). TILT-321 is engineered to replicate only in cancer cells, leading to a high concentration of the aMUC1aCD3 molecule in the tumor microenvironment. Infection and cell viability assays were performed to determine the oncolytic potential of the novel construct. The functionality of the virus-derived aMUC1aCD3 was evaluated invitro. When TILT-321 was combined with allogeneic Tcells, rapid tumor cell lysis was observed. TILT-321-infected cells secreted functional aMUC1aCD3, as shown by increased Tcell activity and its binding to MUC1 and CD3. Invivo, TILT-321 treatment led to effective antitumor efficacy mediated by increased intratumoral Tcell activity in an A549 and patient-derived ovarian cancer xenograft mouse model humanized with peripheral blood mononuclear cells (PBMC). This study provides a proof of concept for an effective strategy to overcome the key limitations of recombinant bispecific Tcell engager delivery for solid tumor treatment.

Outcomes of Tumor Lysis Syndrome in Hospitalized Patients: A Retrospective Cohort Study from National Inpatient Sample 2016-2019

Background: Tumor lysis syndrome (TLS) is an oncologic emergency caused by rapid cell lysis spilling intracellular contents (potassium, phosphate, and nucleic acids) into the systemic circulation leading to hyperkalemia, hyperphosphatemia, secondary hypocalcemia, hyperuricemia, and acute kidney injury. We aim to study outcomes like in-hospital mortality, length of stay (LOS) and associated complications among TLS patients. Methods: This is a retrospective cohort study from TLS hospitalizations between January 1, 2016, and December 31, 2019, using the 2016-2019 National Inpatient Sample (NIS), the largest all-payer public database of hospital care data in the United States. Our study sample included, TLS hospitalizations with age 18 years or older, using the ICD 10 diagnosis codes validated in previous studies. Results: From 2016 to 2019, the incidence rate of TLS was 36 per 100,000 (N=10,277) hospitalizations. Among TLS admissions, 36.9% (n= 3794) were females and mean age was 63±15.5 years (P<0.001). Moreover, 67.5% were Whites, 14.7% were Blacks, 9.4% were Hispanics, and 3.7% were Asians. A total of 2494(47%) hospitalizations had a Charlson Comorbidity index (CCI) of three or higher. TLS hospitalizations were complicated by sepsis (10.6%, N=1089), Cardiac arrest (2.9%, N=298), AKI (68.8%, N=7072), ARDS (0.6%, N=64, requirement for dialysis (8.8%, N=906), GI bleed (5.5%, N=568), Non-traumatic brain hemorrhage (1.4%, N=142), Hyperkalemia (29%, N=2976), Hypocalcemia (10%, N=1042), requirement of mechanical ventilation (11%, N=1130). Mean length of stay was 13.7 days±14.6. Multivariate regression analysis showed that, the predictors for increased in-hospital mortality were GI bleed(aOR: 1.43; 95% CI: 1.16, 1.7; p=0.001), Sepsis (aOR: 2.77; 95% CI: 2.35, 3.25; p<0.001), AKI (aOR: 2.89; 95% CI: 2.49, 3.35; p<0.001), hyperkalemia (aOR: 1.54; 95% CI: 1.37, 1.73; p<0.001) after controlling for age, race ,gender, regional location of the hospital, and income. Females have higher in hospital mortality from TLS compared to males (aOR: 1.22; 95% CI: 1.09, 1.36; p=0.001). From 2016-2019, the annual incidence of TLS per 100,000 hospitalizations has been increasing at 28.6 in 2016, 33.3 in 2017, 38.3 in 2018 and 44.3 in 2019 (p-trend<0.001). Conclusion: Increased incidence of in hospital mortality could be attributed to GI bleed, hyperkalemia, sepsis, AKI. The annual incidence of TLS is rising every year.

Lysostaphin: Engineering and Potentiation toward Better Applications.

Lysostaphin is a potent bacteriolytic enzyme with endopeptidase activity against the common pathogen Staphylococcus aureus. By digesting the pentaglycine crossbridge in the cell wall peptidoglycan of S. aureus including the methicillin-resistant strains, lysostaphin initiates rapid lysis of planktonic and sessile cells (biofilms) and has great potential for use in agriculture, food industries, and pharmaceutical industries. In the past few decades, there have been tremendous efforts in potentiating lysostaphin for better applications in these fields, including engineering of the enzyme for higher potency and lower immunogenicity with longer-lasting effects, formulation and immobilization of the enzyme for higher stability and better durability, and recombinant expression for low-cost industrial production and in situ biocontrol. These achievements are extensively reviewed in this article focusing on applications in disease control, food preservation, surface decontamination, and pathogen detection. In addition, some basic properties of lysostaphin that have been controversial and only elucidated recently are summarized, including the substrate-binding properties, the number of zinc-binding sites, the substrate range, and the cleavage site in the pentaglycine crossbridge. Resistance to lysostaphin is also highlighted with a focus on various mechanisms. This article is concluded with a discussion on the limitations and future perspectives for the actual applications of lysostaphin.

微生物シングルセルゲノム解析技術bit-MAP®を用いたエンドリシン創薬技術開発

The emergence of antimicrobial-resistant (AMR) bacteria poses an imminent threat to our society and requires urgent actions. We here propose that a bacteriophage-derived protein called endolysin can be exploited as a new antimicrobial agent to tackle this global public heath issue. Endolysins are hydrolytic enzymes that cleave bacterial cell-walls (peptidoglycan layers), allowing for rapid cell lysis in a matter of minutes. Not only this swift response makes a stark contrast with conventional antibiotics, but the protein nature of endolysins makes them amenable to modifications. For example, modular domains of endolysin can be recombined to create new artificial endolysins. Using in-house high-throughput screening system, we show that we can quickly discover multiple endolysins against methicillin-resistant Staphylococcus aureus (MRSA) with improved activity compared to previously reported ones. We also discuss the prospects for the development of antimicrobial endolysins against any potential pathogens using our proprietary microbial single-cell genome sequencing platform, bit-MAP(R).

Emergency Use of Targeted Osmotic Lysis for the Treatment of a Patient with Aggressive Late-Stage Squamous Cell Carcinoma of the Cervix

Upregulation of voltage-gated sodium channels (VGSCs) and Na+/K+-ATPase (sodium pumps) is common across most malignant carcinomas. Targeted osmotic lysis (TOL) is a developing technology in which the concomitant stimulation of VGSCs and pharmacological blockade of sodium pumps causes rapid selective osmotic lysis of carcinoma cells. This treatment of cervical carcinoma is evidence that TOL is a safe, well-tolerated and effective treatment for aggressive advanced carcinomas that has the potential to extend life without compromising its quality. TOL is likely to have broad application for the treatment of advanced-stage carcinomas.

High Efficiency RNA Extraction From Sperm Cells Using Guanidinium Thiocyanate Supplemented With Tris(2-Carboxyethyl)Phosphine.

The extraction of high-quality ribonucleic acid (RNA) from tissues and cells is a key step in many biological assays. Guanidinium thiocyanate-phenol-chloroform (AGPC) is a widely used and efficient method to obtain pure RNA from most tissues and cells. However, it is not efficient with some cells like sperm cells because they are resistant to chaotropic lysis solutions containing guanidinium thiocyanate such as Buffer RLT+ and Trizol. Here, we show that disulfide bonds are responsible for the chemical resistance of sperm cells to RNA extraction reagents. We show that while β-mercaptoethanol (βME) can increase sperm lysis in Buffer RLT+, it has no effect in Trizol and leaves sperm cells intact. We measured the reduction of disulfide bonds in 2,2′-dithiodipyridine (DTDP) and observed that βME has a pH-dependent activity in chaotropic solutions, suggesting that pH is a limiting factor. We identified tris(2-carboxyethyl)phosphine (TCEP) as an efficient lysis enhancer of AGPC solutions that can retain reducing activity even at acidic pH. Trizol supplemented with TCEP allows the complete and rapid lysis of sperm cells, increasing RNA yield by 100-fold and resulting in RNA with optimal quality for reverse transcription and polymerase chain reaction. Our findings highlight the importance of efficient cell lysis and extraction of various macromolecules for bulk and single-cell assays, and can be applied to other lysis-resistant cells and vesicles, thereby optimizing the amount of required starting material and animals.

Microbial Photoinactivation by Visible Light Results in Limited Loss of Membrane Integrity.

Interest in visible light irradiation as a microbial inactivation method has widely increased due to multiple possible applications. Resistance development is considered unlikely, because of the multi-target mechanism, based on the induction of reactive oxygen species by wavelength specific photosensitizers. However, the affected targets are still not completely identified. We investigated membrane integrity with the fluorescence staining kit LIVE/DEAD® BacLight™ on a Gram positive and a Gram negative bacterial species, irradiating Staphylococcus carnosus and Pseudomonas fluorescens with 405 nm and 450 nm. To exclude the generation of viable but nonculturable (VBNC) bacterial cells, we applied an ATP test, measuring the loss of vitality. Pronounced uptake of propidium iodide was only observed in Pseudomonas fluorescens at 405 nm. Transmission electron micrographs revealed no obvious differences between irradiated samples and controls, especially no indication of an increased bacterial cell lysis could be observed. Based on our results and previous literature, we suggest that visible light photoinactivation does not lead to rapid bacterial cell lysis or disruption. However, functional loss of membrane integrity due to depolarization or inactivation of membrane proteins may occur. Decomposition of the bacterial envelope following cell death might be responsible for observations of intracellular component leakage.

A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrusaustraliasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.

Fully 3D printed fluidic devices with integrated valves and pumps for flow injection analysis.

The use of a PolyJet 3D printer to create a microfluidic device that has integrated valves and pumps is described. The process uses liquid support and stacked printing to result in fully printed devices that are ready to use within minutes of fabrication after minimal post-processing. A unique feature of PolyJet printing is the ability to incorporate several different materials of varying properties into one print. In this work, two commercially available materials were used: a rigid-transparent plastic material (VeroClear) was used to define the channel regions and the bulk of the device, while the pumps/valves were printed in a flexible, rubber-like material (Agilus30). The entire process, from initial design to testing takes less than 4 hours to complete. The performance of the valves and pumps were characterized by fluorescence microscopy. A flow injection analysis device that enabled the discrete injections of analyte plugs was created, with on-chip pumps being used to move the fluid streams. The injection process was found to be reproducible and linearly correlated with changes in analyte concentration. The utility was demonstrated with the injection and rapid lysis of fluorescently-labeled endothelial cells. The ability to produce a device with integrated pumps/valves in one process significantly adds to the applicability of 3D printing to create microfluidic devices for analytical measurements.

Na2S2O8 Nanoparticles Trigger Antitumor Immunotherapy through Reactive Oxygen Species Storm and Surge of Tumor Osmolarity.

Although more attention has been attracted to the therapy based on reactive oxygen species (ROS) for tumor therapy in recent years, such as photodynamic therapy and chemodynamic therapy, the limited ROS production rate leads to their poor treatment effect owing to the relatively low content of O2 and H2O2 in tumor microenvironments, confined light penetration depth, strict Fenton reaction conditions (pH 3-4), and so on. Therefore, it is urgent to explore the new agents with highly efficient ROS generation capacity. Herein, we first prepared phospholipid coated Na2S2O8 nanoparticles (PNSO NPs) as new ROS generation agents for in situ generating Na+ and S2O82- through gradual degradation, which can then be changed to toxic •SO4- (a novel reported ROS) and •OH regardless of the amount of H2O2 and pH value in the tumor microenvironment (TME). As the generation of a large amount of Na+, PNSO NPs can bypass the ion transport rules of cells through endocytosis to deliver large amounts of Na+ into the cells, resulting in a surge of osmolarity and rapid cell rupture and lysis. Osmotic pressure induced by PNSO NPs will further lead to an unusual manner of cell death: caspase-1-related pyroptosis. Moreover, all of above effects will cause high immunogenic cell death, regulate the immunosuppressed TME, and then activate systemic antitumor immune responses to combat tumor metastasis and recurrence. We believe PNSO NPs will be new and potential ROS generation agents, and this work will broaden the thinking of the exploring of new antitumor nanodrugs.

Modulation of Inflammasome and Pyroptosis by Olaparib, a PARP-1 Inhibitor, in the R6/2 Mouse Model of Huntington's Disease.

Pyroptosis is a type of cell death that is caspase-1 (Casp-1) dependent, which leads to a rapid cell lysis, and it is linked to the inflammasome. We recently showed that pyroptotic cell death occurs in Huntington’s disease (HD). Moreover, we previously described the beneficial effects of a PARP-1 inhibitor in HD. In this study, we investigated the neuroprotective effect of Olaparib, an inhibitor of PARP-1, in the mouse model of Huntington’s disease. R6/2 mice were administered Olaparib or vehicle from pre-symptomatic to late stages. Behavioral studies were performed to investigate clinical effects of the compound. Immunohistochemical and Western blotting studies were performed to evaluate neuroprotection and the impact of the compound on the pathway of neuronal death in the HD mice. Our results indicate that Olaparib administration starting from the pre-symptomatic stage of the neurodegenerative disease increased survival, ameliorated the neurological deficits, and improved clinical outcomes in neurobehavioral tests mainly by modulating the inflammasome activation. These results suggest that Olaparib, a commercially available drug already in use as an anti-neoplastic compound, exerts a neuroprotective effect and could be a useful pharmaceutical agent for Huntington’s disease therapy.

Besnoitia besnoiti\u2013driven endothelial host cell cycle alteration

Besnoitia besnoiti is an important obligate intracellular parasite of cattle which primarily infects host endothelial cells of blood vessels during the acute phase of infection. Similar to the closely related parasite Toxoplasma gondii, B. besnoiti has fast proliferating properties leading to rapid host cell lysis within 24–30 h p.i. in vitro. Some apicomplexan parasites were demonstrated to modulate the host cellular cell cycle to successfully perform their intracellular development. As such, we recently demonstrated that T. gondii tachyzoites induce G2/M arrest accompanied by chromosome missegregation, cell spindle alteration, formation of supernumerary centrosomes, and cytokinesis impairment when infecting primary bovine umbilical vein endothelial cells (BUVEC). Here, we follow a comparative approach by using the same host endothelial cell system for B. besnoiti infections. The current data showed that—in terms of host cell cycle modulation—infections of BUVEC by B. besnoiti tachyzoites indeed differ significantly from those by T. gondii. As such, cyclin expression patterns demonstrated a significant upregulation of cyclin E1 in B. besnoiti–infected BUVEC, thereby indicating parasite-driven host cell stasis at G1-to-S phase transition. In line, the mitotic phase of host cell cycle was not influenced since alterations of chromosome segregation, mitotic spindle formation, and cytokinesis were not observed. In contrast to respective T. gondii–related data, we furthermore found a significant upregulation of histone H3 (S10) phosphorylation in B. besnoiti–infected BUVEC, thereby indicating enhanced chromosome condensation to occur in these cells. In line to altered G1/S-transition, we here additionally showed that subcellular abundance of proliferating cell nuclear antigen (PCNA), a marker for G1 and S phase sub-stages, was affected by B. besnoiti since infected cells showed increased nuclear PCNA levels when compared with that of control cells.