Synthetic liposomes are widely used as drug delivery vehicles in biomedical treatments, such as for mRNA-based antiviral vaccines like those recently developed against SARS-CoV-2. Extracellular vesicles (EVs), which are naturally produced by cells, have emerged as a next-generation delivery system. However, key questions regarding their origin within cells remain unresolved. In this regard, plasma membrane vesicles (PMVs), which are essentially produced from the cellular plasma membrane (PM), present a promising alternative. Unfortunately, their properties relevant to biomedical applications have not be extensively studied. Therefore, we conducted a thorough investigation of the methods used in the production of PMVs. By leveraging advanced fluorescence techniques in microscopy and flow cytometry, we demonstrated a strong dependence of the physicochemical attributes of PMVs on the chemicals used during their production. Following established protocols employing chemicals such as paraformaldehyde (PFA), N-ethylmaleimide (NEM) or dl-dithiothreitol (DTT) and by developing a modified NEM-based method that involved a hypotonic shock step, we generated PMVs from THP-1 CD1d cells. We systematically compared key parameters such as vesicle output, their size distribution, vesicular content analysis, vesicular membrane lipid organization and the mobility of a transmembrane protein. Our results revealed distinct trends: PMVs isolated using NEM-based protocols closely resembled natural vesicles, whereas PFA induced significant molecular cross-linking, leading to notable changes in the biophysical properties of the vesicles. Furthermore, our novel NEM protocol enhanced the efficiency of PMV production. In conclusion, our study highlights the unique characteristics of chemically produced PMVs and offers insights into their potentially diverse yet valuable biological functions.

Soybean (Glycine max [L.] Merr.) is a valuable oil crop but is also highly susceptible to environmental stress. Thus, developing approaches to enhance soybean stress resistance is vital to soybean yield improvement. In previous studies, transcription factor Alfin has been shown to serve as an epigenetic regulator of plant growth and development. However, no studies on Alfin have yet been reported in soybean. In this study, the endoplasmic reticulum (ER) stress- and reactive oxygen species (ROS)-related GmAlfin09 was identified. Screening of genes co-expressed with GmAlfin09 unexpectedly led to the identification of soybean peroxidase 6 (GmPRDX6). Further analyses revealed that both GmAlfin09 and GmPRDX6 were responsive to ER stress, with GmPRDX6 localizing to the ER under stress. Promoter binding experiments confirmed the ability of GmAlfin09 to bind to the GmPRDX6 promoter directly. When GmAlfin09 and GmPRDX6 were overexpressed in soybean, enhanced ER stress resistance and decreased ROS levels were observed. Together, these findings suggest that GmAlfin09 promotes the upregulation of GmPRDX6, and GmPRDX6 subsequently localizes to the ER, reduces ROS levels, promotes ER homeostasis, and ensures the normal growth of soybean even under ER stress. This study highlights a vital target gene for future molecular breeding of stress-resistant soybean lines.

Certain therapies that target CD markers on some blood cells can affect pretransfusion testing. Key examples are anti-CD38, CD47 monoclonal antibody (mAb) therapies such as daratumumab (DARA) and magrolimab, which have presented a challenge for transfusion medicine laboratories around the globe. Scientists have been faced with not only introducing a protocol to provide safe blood to patients but also investigating the most effective method to remove the pretransfusion pan-agglutinating interference caused. A number of papers in the last 5 years have reported on various methods to remove pretransfusion interference; however, most of these studies have been conducted only in a few countries. Most recent reviews on this topic have focused on techniques and reagents to remove pretransfusion interference, and dithiothreitol is currently the gold standard for removing DARA interference. However, it was clear from this review that while many laboratories have developed processes for addressing interference in pretransfusion testing, and DARA interference may not be a major issue, there are still laboratories around the world, that may not have adequately addressed this issue. In addition, the impact of mAb interference on widely used techniques such as flow cytometry is unclear.

Critically ill pediatric patients supported on ventricular assist devices (VADs) are increasingly being anticoagulated on bivalirudin, but with difficulty monitoring anticoagulation. Activated partial thromboplastin time (aPTT) has recently been shown to poorly correlate with bivalirudin plasma concentrations, while dTT had excellent correlation. However, aPTT is the more common monitoring test and dTT testing is rarely used. In addition, effects of frequent clinical VAD scenarios (such as inflammation) on the accuracy of aPTT and dTT testing remains uncertain. We reviewed the effects of clinical scenarios (infection/inflammation, chylothorax, and steroids administration) on anticoagulation monitoring in 10 pediatric VAD patients less than 3 years at Cincinnati Children\'s Hospital Medical Center from 10/27/2020 to 5/6/2022 using bivalirudin for anticoagulation. There were 16 inflammation/infection, 3 chylothorax, and 6 steroids events. Correlation between dTT and aPTT was significantly lower after infection/inflammation, with dTT increasing prior to inflammation/infection while aPTT remained unchanged. In addition, steroids are administered to VAD patients to reduce inflammation and thus additionally stabilize anticoagulation. However, this anticoagulation stabilization effect was reflected more accurately by dTT compared to aPTT. In children requiring VAD support utilizing bivalirudin anticoagulation, inflammation/infection is a common occurrence resulting in anticoagulation changes that may be more accurately reflected by dTT as opposed to aPTT.

A positive flow cytometry crossmatch (FCXM) due to donor specific antibodies (DSA) constitutes a risk for kidney transplantation; such a finding may indicates an unacceptable donor for this patient. However, positive FCXM in the absence of DSA is considered discordant and need further investigations. During COVID-19 pandemic, we observed 22% discordant results out of 445 FCXM performed during eight months period in our laboratory and another 7% were invalid due to high background negative control (NC). No study has addressed the impact of COVID-19 pandemic on FCXM and the overall pre-kidney transplant workups or described a solution to deal with these non-specific reactivities. Herein, we analyzed all FCXM results in SARS-CoV-2 seropositive patients and addressed how this pandemic affected significantly the pre-kidney transplant workups, highlighting both technical and financial implications. We also shared our modified FCXM procedures using dithiotheritol (DTT) sera treatment or blocking donor cells with negative control human serum (NCS) which we found to be successful to abrogate 98% of all discordant FCXM results and to validate all invalid results due to high background NC. In conclusion, COVID-19 pandemic has affected our HLA laboratory significantly by creating many false positive or invalid crossmatch results. Transplant laboratories must consider this before test interpretations and immune risk assessments. We recommend the use of DTT serum treatment to remove nonspecific bindings in the sera of kidney transplant candidates and the use of NCS-blocked donor cells to correct high background when performing FCXM in transplant candidates or donors with recent history of SARS-CoV-2 immunization respectively.

The patch clamp method is a widely applied electrophysiological technique used to understand ion channel activity and cellular excitation. The formation of a high resistance giga-ohm seal is required to obtain high-quality recordings but can be challenging due to variables including operator experience and cell preparation. Therefore, the identification of methods to promote the formation and longevity of giga-ohm seals may be beneficial. In this report, we describe our observation that the application of reducing agents (DTT and TCEP) to the external bath solution during whole-cell patch clamp recordings of heterologous cells (HEK and LM) and cultured primary cells (DRG neurons) enhanced the success of giga-ohm seal formation. Reducing agents also maintained the integrity of the seal for longer periods of time at strong hyperpolarizing voltages, whereas an oxidizing agent (H2O2) appeared to have the opposite effect. In summary, we report a useful tool to improve the quality of patch clamp recordings that may be helpful in certain experimental contexts.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.

In this study, we developed, optimized, and evaluated in lab and field experiments a wet electrostatic precipitator (ESP) for the collection of ambient PM2.5 (particulate matter with aerodynamic diameter < 2.5 μm) into ultrapure water by applying an electrostatic charge to the particles. We operated the wet ESP at different flow rates and voltages to identify the optimal operating conditions. According to our experimental measurements, a flow rate of 125 lpm and an applied positive voltage of 11 kV resulted in a lower ozone generation of 133 ppb and a particle collection efficiency exceeding 80-90% in all size ranges. For the field tests, the wet ESP was compared with the versatile aerosol concentration enrichment system (VACES) connected to a BioSampler, a PTFE filter sampler, and an OC/EC analyzer (Sunset Laboratory Inc., USA) as a reference. The chemical analysis results indicated the wet ESP concentrations of metal and trace elements were in very good agreement with those measured by the VACES/BioSampler and PTFE filter sampler. Moreover, our results showed comparable total organic carbon (TOC) concentrations measured by the wet ESP, BioSampler, and OC/EC analyzer, while somewhat lower TOC concentrations were measured by the PTFE filter sampler, possibly due to the limitations of extracting water-insoluble organic carbon (WIOC) from a dry substrate in the latter sampler. The comparable TOC content in the wet ESP and BioSampler samples differs from previous findings that showed higher TOC content in BioSampler samples compared to those collected by dry ESP. The results of the Dithiothreitol (DTT) assay showed comparable DTT activity in the VACES/BioSampler and wet ESP PM samples while slightly lower in the PTFE filter samples. Overall, our results suggest that the wet ESP could be a promising alternative to other conventional sampling methods.

Oxidative potential (OP) of fine marine aerosols (PM2.5) over the northern Indian Ocean (N_IO) and equatorial Indian Ocean (E_IO) were studied using shipborne measurements conducted as part of the Integrated Campaign for Aerosols, gases and Radiation Budget (ICARB-2018). During the study, an enhanced concentration of PM2.5 was found over N_IO (27.22 ± 14.29 μg.m-3) compared with E_IO (15.91 ± 2.58 μg.m-3), as N_IO experiences continental outflow from anthropogenically dominated South Asian region. However, E_IO received pristine air masses from the middle of the Arabian Sea, implying a reduced concentration. The OP of PM2.5 was evaluated using a dithiothreitol (DTT) assay. The mass (DTTm or intrinsic OP) and volume (DTTv or extrinsic OP) normalized DTT exhibited a significant spatial variation over the Indian Ocean (IO). Intrinsic OP showed ∼2 times higher values over N_IO than E_IO, indicating aging of aerosols during long-range transport impacts OP of marine aerosol. Similarly, increased concentrations of anthropogenic species such as non-sea sulfate (nssSO42-), nitrate (NO3-), ammonium (NH4+), non-sea potassium ion (nssK+), water-soluble transition metals (Fe, Ti, Zn, Cu, Mn, Cr), elemental carbon (EC), organic carbon (OC), water-soluble organic carbon (WSOC), were also observed over N_IO compared with E_IO. Pearson correlation and multiple linear regression (MLR) analysis revealed that combustion sources, chemical processing and co-transportation of anthropogenic species during long-range transport are the main drivers of intrinsic OP in the outflow region.

The eukaryotic membrane vesicles contain specific sets of proteins that determine vesicle function and shuttle with specific destination. Giardia lamblia contains unknown cytosolic vesicles that are related to the identification of a homolog of human myeloid leukemia factor (MLF) named MLF vesicles (MLFVs). Previous studies suggest that MLF also colocalized with two autophagy machineries, FYVE and ATG8-like protein, and that MLFVs are stress-induced compartments for substrates of the proteasome or autophagy in response to rapamycin, MG132, and chloroquine treatment. A mutant protein of cyclin-dependent kinase 2, CDK2m3, was used to understand whether the aberrant proteins are targeted to degradative compratments. Interestingly, MLF was upregulated by CDK2m3 and they both colocalized within the same vesicles. Autophagy is a self-digestion process that is activated to remove damaged proteins for preventing cell death in response to various stresses. Because of the absence of some autophagy machineries, the mechanism of autophagy is unclear in G. lamblia.
In this study, we tested the six autophagosome and stress inducers in mammalian cells, including MG132, rapamycin, chloroquine, nocodazole, DTT, and G418, and found that their treatment increased reactive oxygen species production and vesicle number and level of MLF, FYVE, and ATG8-like protein in G. lamblia. Five stress inducers also increased the CDK2m3 protein levels and vesicles. Using stress inducers and knockdown system for MLF, we identified that stress induction of CDK2m3 was positively regulated by MLF. An autophagosome-reducing agent, 3-methyl adenine, can reduce MLF and CDK2m3 vesicles and proteins. In addition, knockdown of MLF with CRISPR/Cas9 system reduced cell survival upon treatment with stress inducers. Our newly developed complementation system for CRISPR/Cas9 indicated that complementation of MLF restored cell survival in response to stress inducers. Furthermore, human MLF2, like Giardia MLF, can increase cyst wall protein expression and cyst formation in G. lamblia, and it can colocalize with MLFVs and interact with MLF.
Our results suggest that MLF family proteins are functionally conserved in evolution. Our results also suggest an important role of MLF in survival in stress conditions and that MLFVs share similar stress-induced characteristics with autophagy compartments.