Silencing of ATP1A1 attenuates cell membrane disruption by nanosecond electric pulses.

This study explored the role of the Na/K-ATPase (NKA) in membrane permeabilization induced by nanosecond electric pulses. Using CRISPR/Cas9 and shRNA, we silenced the ATP1A1 gene, which encodes α1 NKA subunit in U937 human monocytes. Silencing reduced the rate and the cumulative uptake of YoPro-1 dye after electroporation by 300-ns, 7-10 kV/cm pulses, while ouabain, a specific NKA inhibitor, enhanced YoPro-1 entry. We conclude that the α1 subunit supports the electropermeabilized membrane state, by forming or stabilizing electropores or by hindering repair mechanisms, and this role is independent of NKA's ion pump function.

Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating Damage to Smooth Muscle: An In Vitro Study.

Cancer ablation therapies aim to be efficient while minimizing damage to healthy tissues. Nanosecond pulsed electric field (nsPEF) is a promising ablation modality because of its selectivity against certain cell types and reduced neuromuscular effects. We compared cell killing efficiency by PEF (100 pulses, 200 ns-10 µs duration, 10 Hz) in a panel of human esophageal cells (normal and pre-malignant epithelial and smooth muscle). Normal epithelial cells were less sensitive than the pre-malignant ones to unipolar PEF (15-20% higher LD50, p < 0.05). Smooth muscle cells (SMC) oriented randomly in the electric field were more sensitive, with 30-40% lower LD50 (p < 0.01). Trains of ten, 300-ns pulses at 10 kV/cm caused twofold weaker electroporative uptake of YO-PRO-1 dye in normal epithelial cells than in either pre-malignant cells or in SMC oriented perpendicularly to the field. Aligning SMC with the field reduced the dye uptake fourfold, along with a twofold reduction in Ca2+ transients. A 300-ns pulse induced a twofold smaller transmembrane potential in cells aligned with the field, making them less vulnerable to electroporation. We infer that damage to SMC from nsPEF ablation of esophageal malignancies can be minimized by applying the electric field parallel to the predominant SMC orientation.

seco-Pregnane Glycosides from Australian Caustic Vine (Cynanchum viminale subsp. australe).

Cynanchum viminale subsp. australe, more commonly known as caustic vine, is a leafless succulent that grows in the northern arid zone of Australia. Toxicity toward livestock has been reported for this species, along with use in traditional medicine and its potential anticancer activity. Disclosed herein are novel seco-pregnane aglycones cynavimigenin A (5) and cynaviminoside A (6), together with new pregnane glycosides cynaviminoside B (7) and cynavimigenin B (8). Cynavimigenin B (8) contains an unprecedented 7-oxobicyclo[2.2.1]heptane moiety in the seco-pregnane series, likely arising from a pinacol-type rearrangement. Interestingly, these isolates displayed only limited cytotoxicity in cancer and normal human cell lines, in addition to low activity against acetylcholinesterase and Sarcoptes scabiei bioassays, suggesting that 5-8 are not associated with the reported toxicity of this plant species.

Urine protects urothelial cells against killing with nanosecond pulsed electric fields.

The quest for safe and effective ablation resulted in the development of nanosecond pulsed electric fields (nsPEF) technology for tumor treatment. For future applications of nsPEF in urothelial cancer treatment, we evaluated the effect of urine presence at the ablation site. We prepared artificial urine (AU) with compounds commonly present in the healthy human urine at physiological concentrations. We compared nsPEF cytotoxicity for cancerous (T24) and non-cancerous (SV-HUC-1) human urothelial cell lines pulsed either in the AU or in a physiological solution (PS). Cell monolayers were exposed to trains of 300-ns, 10-Hz pulses using a two-needle electrode assembly placed orthogonal to the monolayer. The assembly produced the electric field gradually weakening with the distance from the electrodes. The electric field which killed 50 % of cells (LD50) was measured by staining with propidium iodide and matching the stained area with the simulated electric field strength. nsPEF exposure in PS was more cytotoxic to cancer cells. The AU protected both healthy and cancer urothelial cells, increasing their LD50 1.4 and 1.6 times, respectively. Omitting urea from the AU reduced the LD50 for healthy and cancer urothelial cells. Testing the role of other AU components, we found that it was the high concentration of phosphates what also rendered the protective effect of the AU. Our findings suggest that the nsPEF ablation of bladder cancer will be less efficient if the bladder is filled with urine.

Action spectra and mechanisms of (in) efficiency of bipolar electric pulses at electroporation.

The reversal of the electric field direction inhibits various biological effects of nanosecond electric pulses (nsEP). This feature, known as "bipolar cancellation," enables interference targeting of nsEP bioeffects remotely from stimulating electrodes, for prospective applications such as precise cancer ablation and non-invasive deep brain stimulation. This study was undertaken to achieve the maximum cancellation of electroporation, by quantifying the impact of the pulse shape, duration, number, and repetition rate across a broad range of electric field strengths. Monolayers of endothelial cells (BPAE) were electroporated in a non-uniform electric field. Cell membrane permeabilization was quantified by YO-PRO-1 (YP) dye uptake and correlated to local electric field strength. For most conditions tested, adding an opposite polarity phase reduced YP uptake by 50-80 %. The strongest cancellation, which reduced YP uptake by 95-97 %, was accomplished by adding a 50 % second phase to 600-ns pulses delivered at a high repetition rate of 833 kHz. Strobe photography of nanosecond kinetics of membrane potential in single CHO cells revealed the temporal summation of polarization by individual unipolar nsEP applied at sub-MHz rate, leading to enhanced electroporation. In contrast, there was no summation for bipolar pulses, and increasing their repetition rate suppressed electroporation. These new findings are discussed in the context of bipolar cancellation mechanisms and remote focusing applications.

Crosstalk between G-Quadruplexes and Dnmt3a-Mediated Methylation of the c-MYC Oncogene Promoter.

The methylation of cytosines at CpG sites in DNA, carried out de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the c-MYC oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: a c-MYC G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical c-MYC G4. The topology and thermal stability of G4 structures in these DNA models were analyzed using physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing c-MYC G4, resulting in inhibition of its methylation activity. c-MYC G4 formation in a double-stranded context significantly reduces Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of c-MYC transcription.

Impact of G-Quadruplex Structures on Methylation of Model Substrates by DNA Methyltransferase Dnmt3a.

In mammals, de novo methylation of cytosines in DNA CpG sites is performed by DNA methyltransferase Dnmt3a. Changes in the methylation status of CpG islands are critical for gene regulation and for the progression of some cancers. Recently, the potential involvement of DNA G-quadruplexes (G4s) in methylation control has been found. Here, we provide evidence for a link between G4 formation and the function of murine DNA methyltransferase Dnmt3a and its individual domains. As DNA models, we used (i) an isolated G4 formed by oligonucleotide capable of folding into parallel quadruplex and (ii) the same G4 inserted into a double-stranded DNA bearing several CpG sites. Using electrophoretic mobility shift and fluorescence polarization assays, we showed that the Dnmt3a catalytic domain (Dnmt3a-CD), in contrast to regulatory PWWP domain, effectively binds the G4 structure formed in both DNA models. The G4-forming oligonucleotide displaced the DNA substrate from its complex with Dnmt3a-CD, resulting in a dramatic suppression of the enzyme activity. In addition, a direct impact of G4 inserted into the DNA duplex on the methylation of a specific CpG site was revealed. Possible mechanisms of G4-mediated epigenetic regulation may include Dnmt3a sequestration at G4 and/or disruption of Dnmt3a oligomerization on the DNA surface.

Electroporation and cell killing by milli- to nanosecond pulses and avoiding neuromuscular stimulation in cancer ablation.

Ablation therapies aim at eradication of tumors with minimal impact on surrounding healthy tissues. Conventional pulsed electric field (PEF) treatments cause pain and muscle contractions far beyond the ablation area. The ongoing quest is to identify PEF parameters efficient at ablation but not at stimulation. We measured electroporation and cell killing thresholds for 150 ns-1 ms PEF, uni- and bipolar, delivered in 10- to 300-pulse trains at up to 1 MHz rates. Monolayers of murine colon carcinoma cells exposed to PEF were stained with YO-PRO-1 dye to detect electroporation. In 2-4 h, dead cells were labeled with propidium. Electroporation and cell death thresholds determined by matching the stained areas to the electric field intensity were compared to nerve excitation thresholds (Kim et al. in Int J Mol Sci 22(13):7051, 2021). The minimum fourfold ratio of cell killing and stimulation thresholds was achieved with bipolar nanosecond PEF (nsPEF), a sheer benefit over a 500-fold ratio for conventional 100-µs PEF. Increasing the bipolar nsPEF frequency up to 100 kHz within 10-pulse bursts increased ablation thresholds by < 20%. Restricting such bursts to the refractory period after nerve excitation will minimize the number of neuromuscular reactions while maintaining the ablation efficiency and avoiding heating.

TERT rs2853669 as a predictor for overall survival in patients with acute myeloid leukaemia.

INTRODUCTION: the aim of the study was to investigate the contribution of TERT rs2736100 and rs2853669 gene polymorphisms in defining the genetic predisposition to acute myeloid leukaemia (AML), their association with different prognostic markers, and their impact on survival, outcome, and the prognosis of affected patients. Also, we investigated the association of TERT SNPs in AML in the presence or absence of DNMT3A (R882), NPM1, and FLT3 mutations. MATERIAL AND METHODS: A total of 509 participants were enrolled in our study, consisting of 146 AML patients and 363 healthy participants, with no history of malignancy. TERT rs2736100 and rs2853669 polymorphisms were genotyped by using TaqMan SNP genotyping assay FLT3 (ITD, D835), DNMT3A (R882), and NPM1 c.863_864insTCTG (type A) mutations were analised in each AML case. RESULTS: TERT rs2736100 and rs2853669 were not associated with AML risk in the codominant, dominant, recessive, or allelic models. Multivariate Cox regression showed that TERT rs2853669 was a significant predictor for overall survival in AML patients. After adjusting for age, gender, cytogenetic risk group, ECOG status, FLT3, DNMT3A, NPM1 mutation, AML subtype, and treatment, the estimated adjusted hazard ratio (HR adjusted = 1.54, 95% CI: 1.01-2.35) showed that the TERT rs2853669 variant genotype had a negative influence on survival time. CONCLUSIONS: TERT rs2853669 and rs2736100 polymorphisms were not risk factors for developing AML in the Romanian population, but the TERT rs2853669 variant genotype had a negative effect on AML patients' overall survival in the presence of other known prognostic factors.

An open, non-randomised, phase 1/2 trial on the safety, tolerability, and immunogenicity of single-dose vaccine "Sputnik Light" for prevention of coronavirus infection in healthy adults.

BACKGROUND: While the world is experiencing another wave of COVID-19 pandemic, global vaccination program is hampered by an evident shortage in the supply of licensed vaccines. In an effort to satisfy vaccine demands we developed a new single-dose vaccine based on recombinant adenovirus type 26 (rAd26) vector carrying the gene for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein - "Sputnik Light". METHODS: We conducted an open label, prospective, non-randomised phase 1/2 trial aimed to assess safety, tolerability, and immunogenicity of "Sputnik Light" vaccine in a single center in Russia. Primary outcome measures were antigen-specific humoral immunity (Anti-RBD-SARS-CoV-2 antibodies measured by ELISA on days 1, 10, 28, and 42) and safety (number of participants with adverse events monitored throughout the study). Secondary outcome measures were antigen-specific cellular immunity (measured by antigen-dependent CD4+ and CD8+ T-cell proliferation, number of antigen-specific interferon-γ-producing cells as well as interferon-γ concentration upon antigen restimulation) and change in neutralizing antibodies (measured in SARS-CoV-2 neutralization assay). FINDINGS: Most of the solicited adverse reactions were mild (66·4% from all vaccinees), few were moderate (5·5%). No serious adverse events were detected. Assessment of Anti-RBD-SARS-CoV-2 antibodies revealed a group with pre-existing immunity to SARS-CoV-2. Upon this finding we separated all safety and immunogenicity data based on pre-existing immunity to SARS-CoV-2. There were notable differences in the vaccine effects on immunogenicity by the groups. Vaccination of seropositive (N=14) volunteers rapidly boosted RBD-specific IgGs from reciprocal geometric mean titer (​GMT) 594·4 at a baseline up to 26899 comparing to 29·09 in seronegative group (N=96) by day 10. By day 42 seroconversion rate reached 100% (93/93) in seronegative group with GMT 1648. At the same time, in the seropositive group, seroconversion rate by day 42 was 92·9% (13/14) with GMT 19986. Analysis of neutralizing antibodies to SARS-CoV-2 showed 81·7% (76/93) and 92·9% (13/14) seroconversion rates by day 42 with median reciprocal GMT 15·18 and 579·7 in the seronegative and seropositive groups, respectively. Antigen-specific T cell proliferation, formation of IFNy-producing cells, and IFNy secretion were observed in 96·7% (26/27), 96% (24/25), and 96% (24/25) of the seronegative group respectively and in 100% (3/3), 100% (5/5), and 100% (5/5) of the seropositive vaccinees, respectively. INTERPRETATION: The single-dose rAd26 vector-based COVID-19 vaccine "Sputnik Light" has a good safety profile and induces a strong humoral and cellular immune responses both in seronegative and seropositive participants. FUNDING: Russian Direct Investment Fund.

Current Paradigm of Hepatitis E Virus Among Pediatric and Adult Patients.

Hepatitis E virus (HEV) infection is a polymorphic condition, present throughout the world and involving children and adults. Multiple studies over the last decade have contributed to a better understanding of the natural evolution of this infection in various population groups, several reservoirs and transmission routes being identified. To date, acute or chronic HEV-induced hepatitis has in some cases remained underdiagnosed due to the lower accuracy of serological tests and due to the evolutionary possibility with extrahepatic manifestations. Implementation of diagnostic tests based on nucleic acid analysis has increased the detection rate of this disease. The epidemiological and clinical features of HEV hepatitis differ depending on the geographical areas studied. HEV infection is usually a self-limiting condition in immunocompetent patients, but in certain categories of vulnerable patients it can induce a sudden evolution toward acute liver failure (pregnant women) or chronicity (immunosuppressed patients, post-transplant, hematological, or malignant diseases). In acute HEV infections in most cases supportive treatment is sufficient. In patients who develop chronic hepatitis with HEV, dose reduction of immunosuppressive medication should be the first therapeutic step, especially in patients with transplant. In case of unfavorable response, the initiation of antiviral therapy is recommended. In this review, the authors summarized the essential published data related to the epidemiological, clinical, paraclinical, and therapeutic aspects of HEV infection in adult and pediatric patients.

Peculiarities of Neurostimulation by Intense Nanosecond Pulsed Electric Fields: How to Avoid Firing in Peripheral Nerve Fibers.

Intense pulsed electric fields (PEF) are a novel modality for the efficient and targeted ablation of tumors by electroporation. The major adverse side effects of PEF therapies are strong involuntary muscle contractions and pain. Nanosecond-range PEF (nsPEF) are less efficient at neurostimulation and can be employed to minimize such side effects. We quantified the impact of the electrode configuration, PEF strength (up to 20 kV/cm), repetition rate (up to 3 MHz), bi- and triphasic pulse shapes, and pulse duration (down to 10 ns) on eliciting compound action potentials (CAPs) in nerve fibers. The excitation thresholds for single unipolar but not bipolar stimuli followed the classic strength-duration dependence. The addition of the opposite polarity phase for nsPEF increased the excitation threshold, with symmetrical bipolar nsPEF being the least efficient. Stimulation by nsPEF bursts decreased the excitation threshold as a power function above a critical duty cycle of 0.1%. The threshold reduction was much weaker for symmetrical bipolar nsPEF. Supramaximal stimulation by high-rate nsPEF bursts elicited only a single CAP as long as the burst duration did not exceed the nerve refractory period. Such brief bursts of bipolar nsPEF could be the best choice to minimize neuromuscular stimulation in ablation therapies.

Interference targeting of bipolar nanosecond electric pulses for spatially focused electroporation, electrostimulation, and tissue ablation.

Stimulation and electroporation by nanosecond electric pulses (nsEP) are distinguished by a phenomenon of bipolar cancellation, which stands for a reduced efficiency of bipolar pulses compared to unipolar ones. When two pairs of stimulating electrodes are arrayed in a quadrupole, bipolar cancellation inhibits nsEP effects near the electrodes, where the electric field is the strongest. Two properly shaped and synchronized bipolar nsEP overlay into a unipolar pulse towards the center of the electrode array, thus canceling the bipolar cancellation (a "CANCAN effect"). High efficiency of the re-created unipolar nsEP outweighs the weakening of the electric field with distance and focuses nsEP effects to the center. In monolayers of CHO, BPAE, and HEK cells, CANCAN effect achieved by the interference of two bipolar nsEP enhanced electroporation up to tenfold, with a peak at the quadrupole center. Introducing a time interval between bipolar nsEP prevented the formation of a unipolar pulse and eliminated the CANCAN effect. Strong electroporation by CANCAN stimuli killed cells over the entire area encompassed by the electrodes, whereas the time-separated pulses caused ablation only in the strongest electric field near the electrodes. The CANCAN approach is promising for uniform tumor ablation and stimulation targeting away from electrodes.

The role of ESCRT-III and Annexin V in the repair of cell membrane permeabilization by the nanosecond pulsed electric field.

Exposure of living cells to intense nanosecond pulsed electric field (nsPEF) increases membrane permeability to small solutes, presumably by the formation of nanometer-size membrane lesions. Mechanisms responsible for the restoration of membrane integrity over the course of minutes after nsPEF have not been identified. This study explored if ESCRT-III and Annexin V calcium-dependent repair mechanisms, which play critical role in resealing large membrane lesions, are also activated by electroporation and contribute to the membrane resealing. The extent of membrane damage and the time course of resealing were monitored by the time-lapse imaging of propidium (Pr) uptake in human cervical carcinoma (HeLa) cells exposed to trains of 300-ns PEF. The removal of the extracellular Ca2+ slowed down the resealing, although did not prevent it. Recruitment of CHMP4B protein, a component of ESCRT-III complex, to the electroporated plasma membrane was not observed, thus providing no evidence for possible contribution of the macro-vesicle shedding mechanism. In contrast, silencing the AnxA5 gene impaired resealing and reduced the viability of nsPEF-treated cells. We conclude that Annexin V but not ESCRT-III was involved in the repair of HeLa cells permeabilized by 300-ns stimuli, but it was not the only and perhaps not the main repair mechanism.

Safety and efficacy of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine: an interim analysis of a randomised controlled phase 3 trial in Russia.

BACKGROUND: A heterologous recombinant adenovirus (rAd)-based vaccine, Gam-COVID-Vac (Sputnik V), showed a good safety profile and induced strong humoral and cellular immune responses in participants in phase 1/2 clinical trials. Here, we report preliminary results on the efficacy and safety of Gam-COVID-Vac from the interim analysis of this phase 3 trial. METHODS: We did a randomised, double-blind, placebo-controlled, phase 3 trial at 25 hospitals and polyclinics in Moscow, Russia. We included participants aged at least 18 years, with negative SARS-CoV-2 PCR and IgG and IgM tests, no infectious diseases in the 14 days before enrolment, and no other vaccinations in the 30 days before enrolment. Participants were randomly assigned (3:1) to receive vaccine or placebo, with stratification by age group. Investigators, participants, and all study staff were masked to group assignment. The vaccine was administered (0·5 mL/dose) intramuscularly in a prime-boost regimen: a 21-day interval between the first dose (rAd26) and the second dose (rAd5), both vectors carrying the gene for the full-length SARS-CoV-2 glycoprotein S. The primary outcome was the proportion of participants with PCR-confirmed COVID-19 from day 21 after receiving the first dose. All analyses excluded participants with protocol violations: the primary outcome was assessed in participants who had received two doses of vaccine or placebo, serious adverse events were assessed in all participants who had received at least one dose at the time of database lock, and rare adverse events were assessed in all participants who had received two doses and for whom all available data were verified in the case report form at the time of database lock. The trial is registered at ClinicalTrials.gov (NCT04530396). FINDINGS: Between Sept 7 and Nov 24, 2020, 21 977 adults were randomly assigned to the vaccine group (n=16 501) or the placebo group (n=5476). 19 866 received two doses of vaccine or placebo and were included in the primary outcome analysis. From 21 days after the first dose of vaccine (the day of dose 2), 16 (0·1%) of 14 964 participants in the vaccine group and 62 (1·3%) of 4902 in the placebo group were confirmed to have COVID-19; vaccine efficacy was 91·6% (95% CI 85·6-95·2). Most reported adverse events were grade 1 (7485 [94·0%] of 7966 total events). 45 (0·3%) of 16 427 participants in the vaccine group and 23 (0·4%) of 5435 participants in the placebo group had serious adverse events; none were considered associated with vaccination, with confirmation from the independent data monitoring committee. Four deaths were reported during the study (three [<0·1%] of 16 427 participants in the vaccine group and one [<0·1%] of 5435 participants in the placebo group), none of which were considered related to the vaccine. INTERPRETATION: This interim analysis of the phase 3 trial of Gam-COVID-Vac showed 91·6% efficacy against COVID-19 and was well tolerated in a large cohort. FUNDING: Moscow City Health Department, Russian Direct Investment Fund, and Sberbank.

[IGG4-related diseases in endocrinology].

Immunoglobulin-G4-related disease (IgG4-RD) is a chronic immunomediated pathology of different organs of local or systemic nature, which has been established as a separate clinical entity in the early 2000s and is characterized by storiform fibroid inflammation of the affected tissues, their increase, and elevated serum immunoglobulin-G4 (IgG4) levels. The most common manifestations of the disease are major salivary and lacrimal gland enlargement, lymphadenopathy and type 1 autoimmune pancreatitis (AIP1), however, other organs may be also involved (the thyroid, eyes, meninges, heart, lungs, kidneys, aorta, upper airways, mesentery, etc.). The effectiveness of treatment of IgG4-RD, as well as other pathological conditions, is also determined by the timely diagnosis. However, the latter is complicated due to the variety of clinical manifestations and rather variable diagnostic criteria. It is necessary to constantly update the evidence-based knowledge and diagnostic algorithms within this pathology in order to overcome the difficulties, and involve immunologists, endocrinologists, pathologists and specialists in other spheres. This review provides information about the etiology, pathogenesis, and current methods of diagnosis and treatment of IgG4-related diseases, as well as examples of some manifestations of IgG4-RD that an endocrinologist may face in practice.

Hydrodynamics of Vortical Gas Jets Coupled to Point-Like Suction.

Vortical jet flows in the Reynolds number (Re) range from 1000 to 3425 and swirl number (S) below 0.5, alone and in combination with suction through a small aperture, are experimentally investigated using optical visualization. Schlieren photography is employed to assess the vortical flow structure and establish the fundamental understanding of the source-to-sink gas-dynamic coupling, including the role played by flow rate, jet diameter, and the separation distance between the gas jet source and the suction sink. Compared to vortex-free jets, vortical jets for Re>2700 with swirl number S>0.27 experience earlier laminar-to-turbulent transition, with resulting rapid growth of the jet boundary. The ability to control growth of the jet expansion and mass and momentum dissipation into the surrounding is demonstrated via use of a coaxially aligned flow suction placed in the path of a jet. When a swirling jet is completely coupled with a flow suction, jet expansion is significantly suppressed. The suction/sink flow rate imposes a limit on the maximum input/source flow rate of gas jet to achieve complete coupling. Furthermore, there is a maximum distance over which effective coupling can occur, and for all Reynolds numbers considered this distance is shorter than the distance at which the jet structure breaks up into turbulent eddies in the absence of a sink.

Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia.

BACKGROUND: We developed a heterologous COVID-19 vaccine consisting of two components, a recombinant adenovirus type 26 (rAd26) vector and a recombinant adenovirus type 5 (rAd5) vector, both carrying the gene for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (rAd26-S and rAd5-S). We aimed to assess the safety and immunogenicity of two formulations (frozen and lyophilised) of this vaccine. METHODS: We did two open, non-randomised phase 1/2 studies at two hospitals in Russia. We enrolled healthy adult volunteers (men and women) aged 18-60 years to both studies. In phase 1 of each study, we administered intramuscularly on day 0 either one dose of rAd26-S or one dose of rAd5-S and assessed the safety of the two components for 28 days. In phase 2 of the study, which began no earlier than 5 days after phase 1 vaccination, we administered intramuscularly a prime-boost vaccination, with rAd26-S given on day 0 and rAd5-S on day 21. Primary outcome measures were antigen-specific humoral immunity (SARS-CoV-2-specific antibodies measured by ELISA on days 0, 14, 21, 28, and 42) and safety (number of participants with adverse events monitored throughout the study). Secondary outcome measures were antigen-specific cellular immunity (T-cell responses and interferon-γ concentration) and change in neutralising antibodies (detected with a SARS-CoV-2 neutralisation assay). These trials are registered with ClinicalTrials.gov, NCT04436471 and NCT04437875. FINDINGS: Between June 18 and Aug 3, 2020, we enrolled 76 participants to the two studies (38 in each study). In each study, nine volunteers received rAd26-S in phase 1, nine received rAd5-S in phase 1, and 20 received rAd26-S and rAd5-S in phase 2. Both vaccine formulations were safe and well tolerated. The most common adverse events were pain at injection site (44 [58%]), hyperthermia (38 [50%]), headache (32 [42%]), asthenia (21 [28%]), and muscle and joint pain (18 [24%]). Most adverse events were mild and no serious adverse events were detected. All participants produced antibodies to SARS-CoV-2 glycoprotein. At day 42, receptor binding domain-specific IgG titres were 14 703 with the frozen formulation and 11 143 with the lyophilised formulation, and neutralising antibodies were 49·25 with the frozen formulation and 45·95 with the lyophilised formulation, with a seroconversion rate of 100%. Cell-mediated responses were detected in all participants at day 28, with median cell proliferation of 2·5% CD4+ and 1·3% CD8+ with the frozen formulation, and a median cell proliferation of 1·3% CD4+ and 1·1% CD8+ with the lyophilised formulation. INTERPRETATION: The heterologous rAd26 and rAd5 vector-based COVID-19 vaccine has a good safety profile and induced strong humoral and cellular immune responses in participants. Further investigation is needed of the effectiveness of this vaccine for prevention of COVID-19. FUNDING: Ministry of Health of the Russian Federation.

Electrohydrodynamics of Gas-Assisted Electrospray Ionization Mass Spectrometry.

Gas-flow assistance is commonly used in ESI-MS for improved transport and desolvation, and fundamental understanding of the underlying phenomena is essential for improvement of aerodynamic interfaces that couple ESI sources and MS. For this purpose, an electrohydrodynamic model is developed for simulation of charged droplet dynamics under the combined effects of gas flow and electric fields with consideration of space charge interactions within the charged aerosol plume. The model is implemented in COMSOL by exploiting a formalism for establishing the droplet trajectories as a sequence of successive droplets ejected at a frequency defined by the electrospray current. The model is used to assess the effect of two distinct flow configurations and compared to the baseline care of electrospray without assist gas. The simulated flows are jet flows oriented coaxially with the ESI spray, with and without imposed vorticity (swirling). Droplet trajectory simulations of a bimodal droplet population consisting of large primary droplets and small progeny droplets reveal a unique capability for vortical assist jet flow to selectively transmit smaller droplets into the MS due to inertial separation. ESI-MS analysis of fluorinated phosphazines subjected to the different gas flow conditions supports the model predictions. The electrohydrodynamic model developed in this work provides a versatile tool to analyze and design aerodynamic ESI interfaces with rigorous incorporation of drag, inertia, and space-charge repulsion and can be used as a powerful simulation methodology for optimizing charged droplet transmission and ultimately improved analytical performance of gas-assisted ESI-MS workflows.

The interplay of excitation and electroporation in nanosecond pulse stimulation.

Conventional electric stimuli of micro- and millisecond duration excite or activate cells at voltages 10-100 times below the electroporation threshold. This ratio is remarkably different for nanosecond electric pulses (nsEP), which caused excitation and activation only at or above the electroporation threshold in diverse cell lines, primary cardiomyocytes, neurons, and chromaffin cells. Depolarization to the excitation threshold often results from (or is assisted by) the loss of the resting membrane potential due to ion leaks across the membrane permeabilized by nsEP. Slow membrane resealing and the build-up of electroporation damages prevent repetitive excitation by nsEP. However, peripheral nerves and muscles are exempt from this rule and withstand multiple cycles of excitation by nsEP without the loss of function or signs of electroporation. We show that the damage-free excitation by nsEP may be enabled by the membrane charging time constant sufficiently large to (1) cap the peak transmembrane voltage during nsEP below the electroporation threshold, and (2) extend the post-nsEP depolarization long enough to activate voltage-gated ion channels. The low excitatory efficacy of nsEP compared to longer pulses makes them advantageous for medical applications where the neuromuscular excitation is an unwanted side effect, such as electroporation-based cancer and tissue ablation.