Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection.

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.

Measurement of Neuropeptide Y Using Aptamer-Modified Microelectrodes by Electrochemical Impedance Spectroscopy.

Aptamer-modified microelectrodes for Neuropeptide Y measurement by electrochemical impedance spectroscopy was described here. The advantages of using carbon fiber or platinum microelectrodes are because they are promising materials with high electrical conductivity, chemical stability, and high surface area that can be easily modified on their surface. The immobilization and biofouling were studied and compared using EIS. Moreover, the adsorption of NPY to the aptamer-modified microelectrodes was also demonstrated by EIS. Changes of -ω*Zimag, an impedance factor that gives information of the capacitance, is directly correlated with concentrations. A widely linear range was obtained from 10 to 1000 ng/mL of NPY. This method was able to detect NPY without performing a redox reaction by adsorption at the surface of the microelectrodes, with the specificity provided by aptamer functionalization of the microelectrode surface.

Naturally processed HLA-DR3-restricted HHV-6B peptides are recognized broadly with polyfunctional and cytotoxic CD4 T-cell responses.

Human herpes virus 6B (HHV-6B) is a widespread virus that infects most people early in infancy and establishes a chronic life-long infection with periodic reactivation. CD4 T cells have been implicated in control of HHV-6B, but antigenic targets and functional characteristics of the CD4 T-cell response are poorly understood. We identified 25 naturally processed MHC-II peptides, derived from six different HHV-6B proteins, and showed that they were recognized by CD4 T-cell responses in HLA-matched donors. The peptides were identified by mass spectrometry after elution from HLA-DR molecules isolated from HHV-6B-infected T cells. The peptides showed strong binding to matched HLA alleles and elicited recall T-cell responses in vitro. T-cell lines expanded in vitro were used for functional characterization of the response. Responding cells were mainly CD3+ CD4+ , produced IFN-γ, TNF-α, and low levels of IL-2, alone or in combination, highlighting the presence of polyfunctional T cells in the overall response. Many of the responding cells mobilized CD107a, stored granzyme B, and mediated specific killing of peptide-pulsed target cells. These results highlight a potential role for polyfunctional cytotoxic CD4 T cells in the long-term control of HHV-6B infection.

Induction of H7N9-Cross-Reactive Antibody-Dependent Cellular Cytotoxicity Antibodies by Human Seasonal Influenza A Viruses that are Directed Toward the Nucleoprotein.

Antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) against avian influenza virus subtypes, including H7N9 and H5N1, have been detected in human sera. Using NK cell activation and NK cytotoxicity assays, we compared ADCC-mediating antibodies (ADCC-Abs) in sera collected from healthy infants, children and adults against H7N9 virus-infected cells and recombinant hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) proteins. High titers of ADCC-Abs against H7N9 virus-infected cells were detected in sera from adults and children but not infants. ADCC-Abs titers directed against H7N9 HA or NA proteins. Further analysis showed that ADCC-Abs titers were significantly higher toward H7N9 NP, as compared with H7N9 HA or NA proteins, and correlated strongly with ADCC-Abs titers against H7N9 virus-infected cells. Indeed, ADCC-Abs to NPs of seasonal H1N1 and H3N2 viruses correlated strongly with ADCC-Abs to H7N9 NP, suggesting that seasonal influenza infections and vaccinations may induce these cross-reactive antibodies. Targeting ADCC-Abs to internal proteins may be a potential mechanism of universal vaccine design.

High Antibody-Dependent Cellular Cytotoxicity Antibody Titers to H5N1 and H7N9 Avian Influenza A Viruses in Healthy US Adults and Older Children.

Human influenza is a highly contagious acute respiratory illness that is responsible for significant morbidity and excess mortality worldwide. In addition to neutralizing antibodies, there are antibodies that bind to influenza virus-infected cells and mediate lysis of the infected cells by natural killer (NK) cells (antibody-dependent cellular cytotoxicity [ADCC]) or complement (complement-dependent lysis [CDL]). We analyzed sera obtained from 16 healthy adults (18-63 years of age), 52 children (2-17 years of age), and 10 infants (0.75-1 year of age) in the United States, who were unlikely to have been exposed to the avian H7N9 subtype of influenza A virus, by ADCC and CDL assays. As expected, none of these sera had detectable levels of hemagglutination-inhibiting antibodies against the H7N9 virus, but we unexpectedly found high titers of ADCC antibodies to the H7N9 subtype virus in all sera from adults and children aged ≥8 years.

CD4+ T cells provide intermolecular help to generate robust antibody responses in vaccinia virus-vaccinated humans.

Immunization with vaccinia virus elicits a protective Ab response that is almost completely CD4(+) T cell dependent. A recent study in a rodent model observed a deterministic linkage between Ab and CD4(+) T cell responses to particular vaccinia virus proteins suggesting that CD4(+) T cell help is preferentially provided to B cells with the same protein specificity (Sette et al. 2008. Immunity 28: 847-858). However, a causal linkage between Ab and CD4(+) T cell responses to vaccinia or any other large pathogen in humans has yet to be done. In this study, we measured the Ab and CD4(+) T cell responses against four vaccinia viral proteins (A27L, A33R, B5R, and L1R) known to be strongly targeted by humoral and cellular responses induced by vaccinia virus vaccination in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors. Our data indicate that there is no direct linkage between Ab and CD4(+) T cell responses against each individual protein in both short-term and long-term immunized donors. Together with the observation that the presence of immune responses to these four proteins is linked together within donors, our data suggest that in vaccinia-immunized humans, individual viral proteins are not the primary recognition unit of CD4(+) T cell help for B cells. Therefore, we have for the first time, to our knowledge, shown evidence that CD4(+) T cells provide intermolecular (also known as noncognate or heterotypic) help to generate robust Ab responses against four vaccinia viral proteins in humans.

Incidental Breast Hemangioma on Breast MRI: A Case Report.

Vascular tumors of the breast are rare, but benign hemangiomas are the most common type. Capillary hemangiomas are a subset of benign vascular tumors that involve smaller vessel sizes. They are difficult to diagnose with mammography and ultrasound, as they lack pathognomonic features and are frequently not seen. MRI is the most sensitive imaging tool. The lesions appear similar to angiosarcoma or ductal carcinoma in situ on imaging, which further complicates the diagnosis. A biopsy of the lesions is required for a definitive diagnosis. In this report, a 49-year-old female with newly diagnosed breast cancer is incidentally found to have a capillary hemangioma on staging breast MRI that was confirmed with a biopsy and excised along with the primary breast cancer with a partial mastectomy. The imaging findings of breast hemangioma on mammography, ultrasound, and MRI are also reviewed and described in this report.

Measurement of neuropeptide Y with molecularly imprinted polypyrrole on carbon fiber microelectrodes.

The measurement of neuropeptides using small electrodes for high spatial resolution would provide us with localized information on the release of neuromolecules. The release of Neuropeptide Y (NPY) is related to different neurological diseases such as stress, obesity, and PTSD, among others. In this conference paper, we electrodeposited polypyrrole on carbon fiber microelectrodes in the presence of NPY to develop a molecularly imprinted polypyrrole sensitive to NPY. Optimization of the electrodeposition process resulted in the full coverage of the polymer with nucleation sites on the carbon fiber ridges, achieving completion by the seventh cycle. Electrodeposition was performed for five cycles, and using cyclic voltammetry (CV), we studied the change in the oxidation current peak for polypyrrole due to the presence of NPY. We also observed a change in capacitance due to the presence of NPY, which was studied by electrochemical impedance spectroscopy (EIS). A linear correlation was found between the oxidation peak and the concentration of NPY between 50 ng/mL and 1000 ng/mL. In addition, a linear correlation was also found between microelectrode capacitance and the concentration of NPY between 50 ng/mL and 1000 ng/mL at 100 kHz.

Au-decorated Sb2Se3 photocathodes for solar-driven CO2 reduction.

Photoelectrodes with FTO/Au/Sb2Se3/TiO2/Au architecture were studied in photoelectrochemical CO2 reduction reaction (PEC CO2RR). The preparation is based on a simple spin coating technique, where nanorod-like structures were obtained for Sb2Se3, as confirmed by SEM images. A thin conformal layer of TiO2 was coated on the Sb2Se3 nanorods via ALD, which acted as both an electron transfer layer and a protective coating. Au nanoparticles were deposited as co-catalysts via photo-assisted electrodeposition at different applied potentials to control their growth and morphology. The use of such architectures has not been explored in CO2RR yet. The photoelectrochemical performance for CO2RR was investigated with different Au catalyst loadings. A photocurrent density of ∼7.5 mA cm-2 at -0.57 V vs. RHE for syngas generation was achieved, with an average Faradaic efficiency of 25 ± 6% for CO and 63 ± 12% for H2. The presented results point toward the use of Sb2Se3-based photoelectrodes in solar CO2 conversion applications.

Perspectives and Behaviors of People with Diabetes toward Time in Range and Glucose Control in Diabetes Management: An Online Survey.

INTRODUCTION: This study assessed experiences, attitudes, and behaviors of people with diabetes (PwD) regarding diabetes self-management and glucose control, and their level of awareness, knowledge, and attitudes toward time in range (TIR). METHODS: This quantitative survey was conducted using an online questionnaire across seven countries. Respondents were PwD classified into three subgroups: type 1 (T1), type 2 insulin (T2 insulin), and type 2 not on insulin (T2 N/insulin). RESULTS: Respondents included 621 people in the T1, 780 people in the T2 insulin, and 735 people in the T2 N/insulin subgroups. Awareness of TIR was low, particularly in the T2 N/insulin subgroup (T1 53%, T2 insulin 29%, T2 N/insulin 9%). Despite a lower current use of continuous glucose monitoring (CGM) among the T2 insulin and T2 N/insulin participants (38% and 9%, respectively), versus T1 participants (64%), most (> 70%) were positive toward utilizing new tools and measures to self-manage blood glucose. Recommendations from their healthcare professionals (HCPs) were cited as a strong motivator to try new measures for analyzing glucose levels. The main barriers cited were limited access to CGM and lack of understanding of TIR benefits. Cost was the main reason given by ≥ 40% of respondents for stopping CGM use. CONCLUSIONS: There is an unmet need in diabetes management, and TIR and CGM offer a potential solution. PwD are motivated to manage their blood glucose levels and are positive toward utilizing new tools and measures to achieve this goal. HCPs play a pivotal role in informing and guiding PwD on new measures for analyzing glucose.

A human CD4+ T cell epitope in the influenza hemagglutinin is cross-reactive to influenza A virus subtypes and to influenza B virus.

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.

Neuroectodermal Diseases: A Comparative Case Report Study.

Neuroectodermal disease involves abnormalities that arise from the ectodermal origin, such as the nervous system, eyeball, retina, and skin. Due to the rarity of the disease, it is often underdiagnosed or misdiagnosed. In this study, the researcher presents two cases of pediatric patients with no fetomaternal complications who presented with focal seizures as their initial complaint. During the examination, varying skin color pigmentation and an abnormal neurophysical examination were observed. Cranial imaging showed hemimegalencephaly and voltage asymmetry on EEG. Skin biopsy was performed on both cases, which revealed basketweave orthokeratosis. The combination of a triad of intractable epilepsy, developmental delay, and cutaneous lesion prompted the consideration of a neuroectodermal disease. The study shows two cases of hypomelanosis of Ito and nevus syndrome, both of which may be due to mTOR and RAS pathways, respectively.

The Predictive Factors of Hospital Bankruptcy-An Exploratory Study.

The United States healthcare industry has witnessed a number of hospitals declare bankruptcy. This has a meaningful impact on local communities with vast implications on access, cost, and quality of care available. In our research, we seek to determine what contemporary structural and operational factors influence a bankruptcy outcome, and craft predictive models to guide healthcare leaders on how to best avoid bankruptcy in the future. In this exploratory study we performed, a single-year cross-sectional analysis of short-term acute care hospitals in the United States and subsequently developed three predictive models: logistic regression, a linear support vector machine (SVM) model with hinge function, and a perceptron neural network. Data sources include Definitive Healthcare and Becker's Hospital Review 2019 report with 3121 observations of 32 variables with 27 observed bankruptcies. The three models consistently indicate that 18 variables have a significant impact on predicting hospital bankruptcy. Currently, there is limited literature concerning financial forecasting models and knowledge detailing the factors associated with hospital bankruptcy. By having tailored knowledge of predictive factors to establish a sound financial structure, healthcare institutions at large can be empowered to take proactive steps to avoid financial distress at the organizational level and ensure long-term financial viability.

Leveraging mHealth for the Treatment and Management of PLHIV.

Objective: The objective of this systematic review was to analyze published literature from the last five years to assess facilitators and barriers to the adoption of mHealth as interventions to treat and manage HIV for PLHIV (people living with HIV). The primary outcomes were physical and mental conditions. The secondary outcomes were behavior based (substance use, care engagement, and healthy habits). Methods: Four databases (PubMed, CINAHL, Web of Science, and ScienceDirect) were queried on 9/2/2022 for peer-reviewed studies on the treatment and management of PLHIV with mHealth as the intervention. The review was conducted in accordance with the Kruse Protocol and reported in accordance with PRISMA 2020. Results: Five mHealth interventions were identified across 32 studies that resulted in improvements in physical health, mental health, care engagement, and behavior change. mHealth interventions offer both convenience and privacy, meet a digital preference, increase health knowledge, decrease healthcare utilization, and increase quality of life. Barriers are cost of technology and incentives, training of staff, security concerns, digital literacy gap, distribution of technology, technical issues, usability, and visual cues are not available over the phone. Conclusion: mHealth offers interventions to improve physical health, mental health, care engagement, and behavior for PLHIV. There are many advantages to this intervention and very few barriers to its adoption. The barriers are strong, however, and should be addressed through policy. Further research should focus on specific apps for younger versus older PLHIV, based on preferences and the digital literacy gap.

Higher Derived Time in Range With IDegLira Versus Insulin Glargine U100 in People With Type 2 Diabetes.

BACKGROUND: Derived time in range (dTIR), calculated from self-monitored blood glucose (SMBG-dTIR) profiles, has demonstrated correlation with risk of cardiovascular and microvascular complications. This post hoc analysis of the DUAL V and DUAL VIII trials aimed to compare dTIR with an insulin degludec/liraglutide fixed-ratio combination (IDegLira) versus insulin glargine 100 units/mL (glargine U100) in people with type 2 diabetes (T2D). MATERIALS AND METHODS: Nine-point SMBG profiles were taken more than 24 hours at baseline and end of trial (EOT: 26 weeks [DUAL V] and 104 weeks [DUAL VIII]) and used to derive the percentage of readings within target range (70-180 mg/dL). Estimated treatment differences (ETDs, IDegLira-glargine U100) were analyzed using analysis of covariance, with treatment as fixed effects and baseline response as a covariate. RESULTS: ETDs for change from baseline to EOT in dTIR were significantly greater with IDegLira versus glargine U100 in DUAL V (4.18%, P = .027) and DUAL VIII (5.17%, P = .001). The proportions of people achieving ≥70% dTIR at EOT with IDegLira and glargine U100, respectively, were 62% and 60% in DUAL V (P = .7541), and 50% and 26% in DUAL VIII (P < .0001). The proportion achieving a ≥5% increase in dTIR from baseline to EOT with IDegLira and glargine U100 was 63% in both groups in DUAL V (P = .9043), and 44% and 25%, respectively, in DUAL VIII (P < .0001). CONCLUSIONS: IDegLira was associated with significantly greater increases in dTIR versus basal insulin alone in people with T2D. TRIAL ID(S): ClinicalTrials.gov, NCT01952145 (DUAL V); ClinicalTrials.gov, NCT02501161 (DUAL VIII).

Antibody titers of individuals vaccinated for COVID-19: A systematic review.

An important preventive measure to mitigate the COVID-19 pandemic is vaccine implementation. In creating vaccines, evoking neutralizing antibody (NAb) production is the main objective. This review determines and compares the NAb titers produced by COVID-19 vaccine recipients based on the vaccine type and the manner of administration. This review includes published articles on studies with healthy participants with a minimum age of 18 years, without previous infections, and those who were given Emergency Use License (EUL) vaccines from WHO. Bias assessment was performed using the Cochrane Risk of Bias and the Newcastle- Ottawa Scale. In all the studies, 40.82% of the primary doses were viral vector platforms. For booster doses, 50% were mRNA platforms. Messenger RNA (mRNA) vaccines have higher titers as homologous than as heterologous vaccines. However, inactivated vaccines and viral vector vaccines have lower titers as homologous than as heterologous vaccines. Meanwhile, subunit vaccines lack data for their titers. Based on the antibody titers, homologous mRNA vaccines are more viral-protective than their heterologous counterparts. Heterologous inactivated and viral vector vaccines are more protective than homologous combinations, mainly when mRNA is the other type in those heterologous combinations. This is because mRNA vaccines elicit higher immunogenicity compared to other types.

Complement-dependent lysis of influenza a virus-infected cells by broadly cross-reactive human monoclonal antibodies.

We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics.

A Newly Engineered A549 Cell Line Expressing ACE2 and TMPRSS2 Is Highly Permissive to SARS-CoV-2, Including the Delta and Omicron Variants.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, causing surges, breakthrough infections, and devastating losses-underscoring the importance of identifying SARS-CoV-2 antivirals. A simple, accessible human cell culture model permissive to SARS-CoV-2 variants is critical for identifying and assessing antivirals in a high-throughput manner. Although human alveolar A549 cells are a valuable model for studying respiratory virus infections, they lack two essential host factors for SARS-CoV-2 infection: angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). SARS-CoV-2 uses the ACE2 receptor for viral entry and TMPRSS2 to prime the SARS-CoV-2 spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a suitable human cell line for SARS-CoV-2 studies by transducing human ACE2 and TMPRSS2 into A549 cells. We show that subclones highly expressing ACE2 and TMPRSS2 ("ACE2plus" and the subclone "ACE2plusC3") are susceptible to infection with SARS-CoV-2, including the delta and omicron variants. These subclones express more ACE2 and TMPRSS2 transcripts than existing commercial A549 cells engineered to express ACE2 and TMPRSS2. Additionally, the antiviral drugs EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir strongly inhibit SARS-CoV-2 variants in our infection model. Our data show that ACE2plusC3 cells are highly permissive to SARS-CoV-2 infection and can be used to identify anti-SARS-CoV-2 drugs.

Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S811-831, with overlapping epitopes presented by common MHC class II proteins HLA-DQ5 and HLA-DP4. These epitopes are recognized by low-abundance CD4 T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. T cell cross-reactivity is driven by the high conservation across human and animal coronaviruses of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC class II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly conserved CD4 T cell epitope broadly recognized across human populations.

Mucosal nanobody IgA as inhalable and affordable prophylactic and therapeutic treatment against SARS-CoV-2 and emerging variants.

Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.