Protection of Omicron Bivalent Vaccine, Previous Infection, and Their Induced Neutralizing Antibodies Against Symptomatic Infection With Omicron XBB.1.16 and EG.5.1.

Background: Data are limited on the protective role of the Omicron BA bivalent vaccine, previous infection, and their induced neutralizing antibodies against Omicron XBB.1.16 and EG.5.1 infection. Methods: We conducted a nested case-control analysis among tertiary hospital staff in Tokyo who had received ≥3 doses of COVID-19 vaccines and donated blood samples in June 2023 (1 month before the Omicron XBB.1.16 and EG.5.1 wave). We identified 206 symptomatic cases between June and September 2023 and selected their controls with 1:1 propensity score matching. We examined the association of vaccination, previous infection, and preinfection live virus neutralizing antibody titers against Omicron XBB.1.16 and EG.5.1 with the risk of COVID-19 infection. Results: Previous infection during the Omicron BA- or XBB-dominant phase was associated with a significantly lower infection risk during the XBB.1.16 and EG.5.1-dominant phase than infection-naive status, with 70% and 100% protection, respectively, whereas Omicron BA bivalent vaccination showed no association. Preinfection neutralizing titers against XBB.1.16 and EG.5.1 were 39% (95% CI, 8%-60%) and 28% (95% CI, 8%-44%) lower in cases than matched controls. Neutralizing activity against XBB.1.16 and EG.5.1 was somewhat detectable in the sera of individuals with previous infection but barely detectable in those who were infection naive and received the Omicron bivalent vaccine. Conclusions: In the era when the Omicron XBB vaccine was unavailable, the Omicron BA bivalent vaccine did not confer the neutralizing activity and protection against Omicron XBB.1.16 and EG.5.1 symptomatic infection. The previous infection afforded neutralizing titers and protection against symptomatic infection with these variants.

Nonhalogen Dry Etching of Metal Carbide TiAlC by Low-Pressure N2/H2 Plasma at Room Temperature.

Ternary metal carbide TiAlC has been proposed as a metal gate material in logic semiconductor devices. It is a hard-to-etch material due to the low volatility of the etch byproducts. Here, a simple, highly controllable, and dry etching method for TiAlC has been first presented using nonhalogen N2/H2 plasmas at low pressure (several Pa) and 20 °C. A capacitively coupled plasma etcher was used to generate N2/H2 plasmas containing active species, such as N, NH, and H to modify the metal carbide surface. The etch rate of TiAlC was obtained at 3 nm/min by using the N2/H2 plasma, whereas no etching occurred with pure N2 plasma or pure H2 plasma under the same conditions. The surface roughness of the TiAlC film etched by N2/H2 plasma was controlled at the atomic level. A smooth etched surface was achieved with a root-mean-square roughness of 0.40 nm, comparable to the initial roughness of 0.44 nm. The plasma properties of the N2/H2 plasmas were diagnosed by using a high-resolution optical emission spectrometer, detecting the NH molecular line at 336 nm. The etching behavior and plasma-surface reaction between N2/H2 plasma and TiAlC were investigated by using in situ spectroscopic ellipsometry, in situ attenuated total reflectance-Fourier transform infrared spectrometry, and X-ray photoelectron spectroscopy. The findings indicate that the N-H, C-N, and Ti(Al)-N bonds form on the TiAlC surface etched by the N2/H2 plasmas. The mechanism for etching of TiAlC involving transformation reactions between inorganic materials (metal carbides) and inorganic etchants (N2/H2 plasma) to form volatile organic compounds such as methylated, methyl-aminated, and aminated metals is proposed. Nonhalogen or nonorganic compound etchants were used during the etching process. The study provides useful insights into microfabrication for large-scale integrated circuits.

Evaluating the Use of Sacran, a Polysaccharide Isolated from Aphanothece sacrum, as a Possible Microbicide for Preventing HIV-1 Infection.

Since combination antiretroviral therapy (cART) was introduced to treat human immunodeficiency virus type-1 (HIV-1)/acquired immunodeficiency syndrome (AIDS), the AIDS mortality rate has markedly decreased, and convalescence in individuals with HIV has improved drastically. However, sexual transmission has made HIV-1 a global epidemic. Sacran is a megamolecular polysaccharide extracted from cyanobacterium Aphanothece sacrum that exhibits numerous desirable characteristics for transdermic applications, such as safety as a biomaterial, a high moisture retention effect, the ability to form a film and hydrogel, and an anti-inflammatory effect. In this study, we evaluated the anti-HIV-1 effects in sacran as a barrier to HIV-1 transmission. Sacran inhibited HIV-1 infection and envelope-dependent cell-to-cell fusion. Moreover, we used a Transwell assay to confirm that sacran inhibited viral diffusion and captured viruses. The synergistic effects of sacran and other anti-HIV infection drugs were also evaluated. HIV-1 infections can be reduced through the synergistic effects of sacran and anti-HIV-1 drugs. Our study suggests using sacran gel to provide protection against HIV-1 transmission.

Acidic sphingomyelinase interactions with lysosomal membranes and cation amphiphilic drugs: A molecular dynamics investigation.

Lysosomes are pivotal in cellular functions and disease, influencing cancer progression and therapy resistance with Acid Sphingomyelinase (ASM) governing their membrane integrity. Moreover, cation amphiphilic drugs (CADs) are known as ASM inhibitors and have anti-cancer activity, but the structural mechanisms of their interactions with the lysosomal membrane and ASM are poorly explored. Our study, leveraging all-atom explicit solvent molecular dynamics simulations, delves into the interaction of glycosylated ASM with the lysosomal membrane and the effects of CAD representatives, i.e., ebastine, hydroxyebastine and loratadine, on the membrane and ASM. Our results confirm the ASM association to the membrane through the saposin domain, previously only shown with coarse-grained models. Furthermore, we elucidated the role of specific residues and ASM-induced membrane curvature in lipid recruitment and orientation. CADs also interfere with the association of ASM with the membrane at the level of a loop in the catalytic domain engaging in membrane interactions. Our computational approach, applicable to various CADs or membrane compositions, provides insights into ASM and CAD interaction with the membrane, offering a valuable tool for future studies.

Deviated binding of anti-HBV nucleoside analog E-CFCP-TP to the reverse transcriptase active site attenuates the effect of drug-resistant mutations.

While certain human hepatitis B virus-targeting nucleoside analogs (NAs) serve as crucial anti-HBV drugs, HBV yet remains to be a major global health threat. E-CFCP is a 4'-modified and fluoromethylenated NA that exhibits potent antiviral activity against both wild-type and drug-resistant HBVs but less potent against human immunodeficiency virus type-1 (HIV-1). Here, we show that HIV-1 with HBV-associated amino acid substitutions introduced into the RT's dNTP-binding site (N-site) is highly susceptible to E-CFCP. We determined the X-ray structures of HBV-associated HIV-1 RT mutants complexed with DNA:E-CFCP-triphosphate (E-CFCP-TP). The structures revealed that exocyclic fluoromethylene pushes the Met184 sidechain backward, and the resultant enlarged hydrophobic pocket accommodates both the fluoromethylene and 4'-cyano moiety of E-CFCP. Structural comparison with the DNA:dGTP/entecavir-triphosphate complex also indicated that the cyclopentene moiety of the bound E-CFCP-TP is slightly skewed and deviated. This positioning partly corresponds to that of the bound dNTP observed in the HIV-1 RT mutant with drug-resistant mutations F160M/M184V, resulting in the attenuation of the structural effects of F160M/M184V substitutions. These results expand our knowledge of the interactions between NAs and the RT N-site and should help further design antiviral NAs against both HIV-1 and HBV.

Brexpiprazole: A new option in treating agitation in Alzheimer's dementia-Insights from transgenic mouse models.

AIM: Brexpiprazole is the first FDA-approved treatment for agitation associated with dementia due to Alzheimer's disease. Agitation in Alzheimer's dementia (AAD) occurs in high prevalence and is a great burden for patients and caregivers. Efficacy, safety, and tolerability of brexpiprazole were demonstrated in the AAD clinical trials. To demonstrate the agitation-ameliorating effect of brexpiprazole in animals, we evaluated brexpiprazole in two AAD mouse models. METHODS: The resident-intruder test was conducted in 5- to 6-month-old Tg2576 mice, given vehicle or brexpiprazole (0.01 or 0.03 mg/kg) orally 1 h before the test. Locomotor activity was measured in 6-month-old APPSL-Tg mice given vehicle or brexpiprazole (0.01 or 0.03 mg/kg) orally the evening before the start of locomotor measurement for 3 days. RESULTS: In the resident-intruder test, Tg2576 mice showed significantly higher attack number and shorter latency to first attack compared to non-Tg mice. In the Tg mice, brexpiprazole treatment (0.03 mg/kg) significantly delayed the latency to first attack and showed a trend toward a decrease in attack number. APPSL-Tg mice (≧6 months old) showed significantly higher locomotion during dark period Phase II (Zeitgeber time [ZT] 16-20) and Phase III (ZT20-24) compared to non-Tg mice, correlating with the clinical observations of late afternoon agitation in Alzheimer's disease. Brexpiprazole treatment (0.01 and 0.03 mg/kg) significantly decreased hyperlocomotion during the Phase III in APPSL-Tg mice. CONCLUSION: The suppression of attack behavior and the reduction of nocturnal hyperlocomotion in these Tg mice may be indicative of the therapeutic effect of brexpiprazole on AAD, as demonstrated in the clinical trials.

Lipid levels correlate with neuronal and dopaminergic markers during the differentiation of SH-SY5Y cells.

Parkinson's Disease (PD) is characterised by the loss of dopaminergic neurons and the deposition of protein inclusions called Lewy Bodies (LBs). LBs are heterogeneous structures composed of protein and lipid molecules and their main constituent is the presynaptic protein α-synuclein. SH-SY5Y cells are neuroblastoma cells commonly used to model PD because they express dopaminergic markers and α-synuclein and they can be differentiated into neuronal cells using established protocols. Despite increasing evidence pointing towards a role of lipids in PD, limited knowledge is available on the lipidome of undifferentiated and differentiated SH-SY5Y cells. Using a combination of lipidomics, proteomics, morphological and electrophysiological measurements, we identified specific lipids, including sphingolipids, whose levels are affected by the differentiation of SH-SY5Y neuroblastoma cells and found that the levels of these lipids correlate with those of neuronal and dopaminergic markers. These results provide a quantitative characterisation of the changes in lipidome associated with the differentiation of SH-SY5Y cells into more neuronal and dopaminergic-like phenotype and serve as a basis for further characterisation of lipid disruptions in association with PD and its risk factors in this dopaminergic-like neuronal cell model.

Discovery of Potent DAG-Lactone Derivatives as HIV Latency Reversing Agents.

Toward human immunodeficiency virus type-1 (HIV-1) cure, cells latently infected with HIV-1 must be eliminated from people living with HIV-1. We previously developed a protein kinase C (PKC) activator, diacylglycerol (DAG)-lactone derivative 3, with high HIV-1 latency-reversing activity, based on YSE028 (2) as a lead compound and found that the activity was correlated with binding affinity for PKC and stability against esterase-mediated hydrolysis. Here, we synthesized new DAG-lactone derivatives not only containing a tertiary ester group or an isoxazole surrogate but also several symmetric alkylidene moieties to improve HIV-1 latency reversing activity. Compound 9a, with a dimethyl group at the α-position of the ester group, exerted twice higher HIV-1 latency reversing activity than compound 3, and compound 26, with the isoxazole moiety, was significantly active. In addition, DAG-lactone derivatives with moderate hydrophobicity and potent biostability showed high biological activity.

HIV-Tocky system to visualize proviral expression dynamics.

Determinants of HIV-1 latency establishment are yet to be elucidated. HIV reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model, HIV-Timer of cell kinetics and activity (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allows, for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies.

HIV Reservoirs and Treatment Strategies toward Curing HIV Infection.

Combination antiretroviral therapy (cART) has significantly improved the prognosis of individuals living with human immunodeficiency virus (HIV). Acquired immunodeficiency syndrome has transformed from a fatal disease to a treatable chronic infection. Currently, effective and safe anti-HIV drugs are available. Although cART can reduce viral production in the body of the patient to below the detection limit, it cannot eliminate the HIV provirus integrated into the host cell genome; hence, the virus will be produced again after cART discontinuation. Therefore, research into a cure (or remission) for HIV has been widely conducted. In this review, we focus on drug development targeting cells latently infected with HIV and assess the progress including our current studies, particularly in terms of the "Shock and Kill", and "Block and Lock" strategies.

Cationic amphiphilic drugs induce accumulation of cytolytic lysoglycerophospholipids in the lysosomes of cancer cells and block their recycling into common membrane glycerophospholipids.

Lysosomes are acidic organelles responsible for lipid catabolism, and their functions can be disrupted by cationic amphiphilic drugs that neutralize lumenal pH and thereby inhibit most lysosomal hydrolases. These drugs can also induce lysosomal membrane permeabilization and cancer cell death, but the underlying mechanism remains elusive. Here, we uncover that the cationic amphiphilic drugs induce a substantial accumulation of cytolytic lysoglycerophospholipids within the lysosomes of cancer cells, and thereby prevent the recycling of lysoglycerophospholipids to produce common membrane glycerophospholipids. Using quantitative mass spectrometry-based shotgun lipidomics, we demonstrate that structurally diverse cationic amphiphilic drugs, along with other types of lysosomal pH-neutralizing reagents, elevate the amounts of lysoglycerophospholipids in MCF7 breast carcinoma cells. Lysoglycerophospholipids constitute ∼11 mol% of total glycerophospholipids in lysosomes purified from MCF7 cells, compared with ∼1 mol% in the cell lysates. Treatment with cationic amphiphilic drug siramesine further elevates the lysosomal lysoglycerophospholipid content to ∼24 mol% of total glycerophospholipids. Exogenously added traceable lysophosphatidylcholine is rapidly acylated to form diacylphosphatidylcholine, but siramesine treatment sequesters the lysophosphatidylcholine in the lysosomes and prevents it from undergoing acylation. These findings shed light on the unexplored role of lysosomes in the recycling of lysoglycerophospholipids and uncover the mechanism of action of promising anticancer agents.

Efficacy and Safety of Treatment with Plasma from COVID-19-Recovered Individuals.

Convalescent plasma therapy, which involves administering plasma from recovered coronavirus disease 2019 (COVID-19) patients to infected individuals, is being explored as a potential treatment for severe cases of COVID-19. This study aims to evaluate the efficacy and safety of convalescent plasma therapy in COVID-19 patients with moderate to severe illness. An open-label, single-arm intervention study was conducted without a control group. Plasma collected from recovered COVID-19 patients was administered to eligible participants. The primary endpoint was the proportion of patients who were placed on artificial ventilation or died within 14 days of transfusion. Secondary endpoints included clinical improvement, viral load measurements, and adverse event monitoring. A total of 59 cases were included in the study. The primary endpoint was evaluated by comparing the rate obtained in the study to an existing rate of 25%. The study also assessed clinical improvement, viral load changes, and safety endpoints through adverse event monitoring. Convalescent plasma therapy shows potential as a treatment option for COVID-19. This study aimed to provide evidence for the efficacy and safety of this therapy and may contribute to its future use in treating severe cases of COVID-19.

Preinfection Neutralizing Antibodies, Omicron BA.5 Breakthrough Infection, and Long COVID: A Propensity Score-Matched Analysis.

BACKGROUND: Data are limited on the role of preinfection humoral immunity protection against Omicron BA.5 infection and long coronavirus disease (COVID) development. METHODS: We conducted nested case-control analysis among tertiary hospital staff in Tokyo who donated blood samples in June 2022 (1 month before Omicron BA.5 wave), approximately 6 months after receiving a third dose of COVID-19 mRNA vaccine. We measured live virus-neutralizing antibody titers against wild type and Omicron BA.5, and anti-receptor-binding domain (RBD) antibody titers at preinfection, and compared them between cases and propensity-matched controls. Among the breakthrough cases, we examined association between preinfection antibody titers and incidence of long COVID. RESULTS: Preinfection anti-RBD and neutralizing antibody titers were lower in cases than controls. Neutralizing titers against wild type and Omicron BA.5 were 64% (95% confidence interval [CI], 42%-77%) and 72% (95% CI, 53%-83%) lower, respectively, in cases than controls. Individuals with previous Omicron BA.1/BA.2 infections were more frequent among controls than cases (10.3% vs 0.8%), and their Omicron BA.5 neutralizing titers were 12.8-fold higher than infection-naive individuals. Among cases, preinfection antibody titers were not associated with incidence of long COVID. CONCLUSIONS: Preinfection immunogenicity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may play a role in protecting against the Omicron BA.5 infection but not preventing long COVID.

Assessing the effects of antiretroviral therapy-latency-reversing agent combination therapy on eradicating replication-competent HIV provirus in a Jurkat cell culture model.

Eradication of HIV-1 latently infected cells is an important issue in HIV treatment. However, there are limited models available to assess therapeutic efficacy in vitro. Here, we present a protocol for establishing a variety of HIV-infected Jurkat cells, including productive and latent status, evaluating the efficacy of antiviral agents, followed by PCR/sequencing-based detection of replication competent HIV provirus. This protocol is useful for optimization of treatment of HIV-1 and provides insights into the mechanisms of clonal selection of heterogeneous HIV-1-infected cells. For complete details on the use and execution of this protocol, please refer to Matsuda et al. (2021).1.

Activation of invasion by oncogenic reprogramming of cholesterol metabolism via increased NPC1 expression and macropinocytosis.

Cancer cells are dependent on cholesterol, and they possess strictly controlled cholesterol homeostasis mechanisms. These allow them to smoothly switch between cholesterol synthesis and uptake to fulfill their needs and to adapt environmental changes. Here we describe a mechanism of how cancer cells employ oncogenic growth factor signaling to promote uptake and utilization of extracellular cholesterol via Myeloid Zinc Finger 1 (MZF1)-mediated Niemann Pick C1 (NPC1) expression and upregulated macropinocytosis. Expression of p95ErbB2, highly oncogenic, standard-treatment resistant form of ErbB2 mobilizes lysosomes and activates EGFR, invasion and macropinocytosis. This is connected to a metabolic shift from cholesterol synthesis to uptake due to macropinocytosis-enabled flow of extracellular cholesterol. NPC1 increase facilitates extracellular cholesterol uptake and is necessary for the invasion of ErbB2 expressing breast cancer spheroids and ovarian cancer organoids, indicating a regulatory role for NPC1 in the process. The ability to obtain cholesterol as a byproduct of increased macropinocytosis allows cancer cells to direct the resources needed for the energy-consuming cholesterol synthesis towards other activities such as invasion. These results demonstrate that macropinocytosis is not only an alternative energy source for cancer cells but also an efficient way to provide building material, such as cholesterol, for its macromolecules and membranes.

Omicron BA.1 neutralizing antibody response following Delta breakthrough infection compared with booster vaccination of BNT162b2.

BACKGROUND: Longitudinal data are lacking to compare booster effects of Delta breakthrough infection versus third vaccine dose on neutralizing antibodies (NAb) against Omicron. METHODS: Participants were the staff of a national research and medical institution in Tokyo who attended serological surveys on June 2021 (baseline) and December 2021 (follow-up); in between, the Delta-dominant epidemic occurred. Of 844 participants who were infection-naïve and had received two doses of BNT162b2 at baseline, we identified 11 breakthrough infections during follow-up. One control matched to each case was selected from boosted and unboosted individuals. We compared live-virus NAb against Wild-type, Delta, and Omicron BA.1 across groups. RESULTS: Breakthrough infection cases showed marked increases in NAb titers against Wild-type (4.1-fold) and Delta (5.5-fold), and 64% had detectable NAb against Omicron BA.1 at follow-up, although the NAb against Omicron after breakthrough infection was 6.7- and 5.2-fold lower than Wild-type and Delta, respectively. The increase was apparent only in symptomatic cases and as high as in the third vaccine recipients. CONCLUSIONS: Symptomatic Delta breakthrough infection increased NAb against Wild-type, Delta, and Omicron BA.1, similar to the third vaccine. Given the much lower NAb against Omicron BA.1, infection prevention measures must be continued irrespective of vaccine and infection history while the immune evasive variants are circulating.

Synthesis and evaluation of DAG-lactone derivatives with HIV-1 latency reversing activity.

Cells latently infected with human immunodeficiency virus type 1 (HIV-1) prevent people living with HIV-1 from obtaining a cure to the infectious disease. Latency reversing agents (LRAs) such as protein kinase C (PKC) activators and histone deacetylase (HDAC) inhibitors can reactivate cells latently infected with HIV-1. Several trials based on treatment with HDAC inhibitors alone, however, failed to reduce the number of latent HIV-1 reservoirs. Herein, we have focused on a diacylglycerol (DAG)-lactone derivative, YSE028 (1), which is a PKC activator with latency reversing activity and no significant cytotoxicity. Caspase-3 activation of YSE028 (1) led to cell apoptosis, specifically in HIV-1 latently infected cells. Structure-activity relationship studies of YSE028 (1) have produced several useful derivatives. Among these, compound 2 is approximately ten times more potent than YSE028 (1) in reactivation of cells latently infected with HIV-1. The activity of DAG-lactone derivatives was correlated with the binding affinity for PKC and the stability against esterase-mediated hydrolysis.

Immunogenicity and safety of single booster dose of KD-414 inactivated COVID-19 vaccine in adults: An open-label, single-center, non-randomized, controlled study in Japan.

Although vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease 2019 (COVID-19) induce effective immune responses, vaccination with booster doses is necessary because of waning immunity. We conducted an open-label, non-randomized, single-arm study in adults in Japan to assess the immunogenicity and safety of a single booster dose of the KD-414 purified whole-SARS-CoV-2-virion inactivated vaccine candidate after vaccination with a primary series of BNT162b2. The primary endpoint was serum neutralizing activity at 7 days after booster injection compared with the primary series of BNT162b2. The SARS-CoV-2-structural protein-binding antibody level and T cell response against SARS-CoV-2-Spike (S) peptides were also examined as secondary endpoints, and safety profile assessments were conducted. Twenty subjects who participated in a previous study declined an injection of KD-414 (non-KD-414 group) and received a booster dose of BNT162b2 instead. The non-KD-414 group was compared to the KD-414 group as a secondary outcome. A single dose of KD-414 induced lower serum neutralizing activity against the wild-type virus within 7 days compared to after the primary series of BNT162b2 but significantly induced anti-SARS-CoV-2-S1-receptor-binding domain-binding immunoglobulin G (IgG) antibodies and SARS-CoV-2-S peptide-specific CD4+ and CD8+ T cell responses. Local or systemic symptoms were significantly lower in the participants who received KD-414 than in those who received BNT162b2 as the third COVID-19 vaccine dose. The present data indicate that a single booster dose of KD-414 induces a substantial immune response in BNT162b2-primed individuals and has a good safety profile, thereby supporting further clinical trials to identify rational targets.

Engineering a membrane-binding protein to trimerize and induce high membrane curvature.

The annexins are a family of Ca2+-dependent peripheral membrane proteins. Several annexins are implicated in plasma membrane repair and are overexpressed in cancer cells. Annexin A4 (ANXA4) and annexin A5 (ANXA5) form trimers that induce high curvature on a membrane surface, a phenomenon deemed to accelerate membrane repair. Despite being highly homologous to ANXA4, annexin A3 (ANXA3) does not form trimers on the membrane surface. Using molecular dynamics simulations, we have reverse engineered an ANXA3-mutant to trimerize on the surface of the membrane and induce high curvature reminiscent of ANXA4. In addition, atomic force microscopy images show that, like ANXA4, the engineered protein forms crystalline arrays on a supported lipid membrane. Despite the trimer-forming and curvature-inducing properties of the engineered ANXA3, it does not accumulate near a membrane lesion in laser-punctured cells and is unable to repair the lesion. Our investigation provides insights into the factors that drive annexin-mediated membrane repair and shows that the membrane-repairing property of trimer-forming annexins also necessitates high membrane binding affinity, other than trimer formation and induction of negative membrane curvature.