Development of thermosensitive hydrogel of Amphotericin-B and Lactoferrin combination-loaded PLGA-PEG-PEI nanoparticles for potential eradication of ocular fungal infections: In-vitro, ex-vivo and in-vivo studies.

The most prevalent conditions among ocular surgery and COVID-19 patients are fungal eye infections, which may cause inflammation and dry eye, and may cause ocular morbidity. Amphotericin-B eye drops are commonly used in the treatment of ocular fungal infections. Lactoferrin is an iron-binding glycoprotein with broad-spectrum antimicrobial activity and is used for the treatment of dry eye, conjunctivitis, and ocular inflammation. However, poor aqueous stability and excessive nasolacrimal duct draining impede these agens' efficiency. The aim of this study was to examine the effect of Amphotericin-B, as an antifungal against Candida albicans, Fusarium, and Aspergillus flavus, and Lactoferrin, as an anti-inflammatory and anti-dry eye, when co-loaded in triblock polymers PLGA-PEG-PEI nanoparticles embedded in P188-P407 ophthalmic thermosensitive gel. The nanoparticles were prepared by a double emulsion solvent evaporation method. The optimized formula showed particle size (177.0 ± 0.3 nm), poly-dispersity index (0.011 ± 0.01), zeta-potential (31.9 ± 0.3 mV), and entrapment% (90.9 ± 0.5) with improved ex-vivo pharmacokinetic parameters and ex-vivo trans-corneal penetrability, compared with drug solution. Confocal laser scanning revealed valuable penetration of fluoro-labeled nanoparticles. Irritation tests (Draize Test), Atomic force microscopy, cell culture and animal tests including histopathological analysis revealed superiority of the nanoparticles in reducing signs of inflammation and eradication of fungal infection in rabbits, without causing any damage to rabbit eyeballs. The nanoparticles exhibited favorable pharmacodynamic features with sustained release profile, and is neither cytotoxic nor irritating in-vitro or in-vivo. The developed formulation might provide a new and safe nanotechnology for treating eye problems, like inflammation and fungal infections.

Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment.

Background & Aims: Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3). Methods: PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining. Results: PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates. Conclusions: Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness. Impact and implications: Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

Inhibition of SARS-CoV-2 nucleocapsid protein-RNA interaction by guanosine oligomeric RNA.

The interaction of the ß-coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein with genomic RNA is initiated by specific RNA regions and subsequently induces the formation of a continuous polymer with characteristic structural units for viral formation. We hypothesized that oligomeric RNAs, whose sequences are absent in the 29.9-kb genome sequence of SARS-CoV-2, might affect RNA-N protein interactions. We identified two such hexameric RNAs, In-1 (CCGGCG) and G6 (GGGGGG), and investigated their effects on the small filamentous/droplet-like structures (< a few µm) of N protein-genomic RNA formed by liquid-liquid phase separation. The small N protein structures were sequence-specifically enhanced by In-1, whereas G6 caused them to coalesce into large droplets. Moreover, we found that a guanosine 12-mer (G12, GGGGGGGGGGGG) expelled preexisting genomic RNA from the small N protein structures. The presence of G12 with the genomic RNA suppressed the formation of the small N protein structures, and alternatively apparently altered phase separation to induce the formation of large droplets with unclear phase boundaries. We showed that the N-terminal RNA-binding domain is required for the stability of the small N protein structures. Our results suggest that G12 may be a strong inhibitor of the RNA-N protein interaction.

Vaccine confidence among those living with allergy during the COVID pandemic (ACCORD): A scoping review.

Background: Reports of allergic reactions to coronavirus disease 2019 (COVID-19) vaccines, coupled with an "infodemic" of misinformation, carry the potential to undermine confidence in the COVID-19 vaccines. However, no attempts have been made to comprehensively synthesize the literature on how allergic disease and fear of allergic reactions to the vaccines contribute to hesitancy. Objectives: Our aim was to review the academic and gray literature on COVID-19 vaccine hesitancy and allergic reactions. Methods: We searched 4 databases (CINAHL, PsycINFO, MEDLINE, and Embase) using a search strategy developed by content and methodologic experts. No restrictions were applied regarding COVID-19 vaccine type, country of study, or patient age. Eligible articles were restricted to 10 languages. Results: Of the 1385 unique records retrieved from our search, 60 articles (4.3%) were included. Allergic reactions to the COVID-19 vaccine were rare but slightly more common in individuals with a history of allergic disease. A fifth of the studies (13 of 60 [22%]) discussed vaccine hesitancy due to possibility of an allergic reaction. Additionally, the present review identified research on details of vaccine-related anaphylaxis (eg, a mean and median [excluding clinical trial data] of 12.4 and 5 cases per million doses, respectively) and allergic reactions (eg, a mean and median [excluding clinical trial data] of 489 and 528 cases per million doses, respectively). Conclusion: COVID-19 vaccine acceptance among individuals living with allergy and among those with no history of allergic disease may be affected by fear of an allergic reaction. Despite the low incidence of allergic reactions to the COVID-19 vaccine, fear of such reactions is one of the most commonly cited concerns reported in the literature.

Impact of process stress on protein stability in highly-loaded solid protein/PEG formulations from small-scale melt extrusion.

As protein-based therapeutics often exhibit a limited stability in liquid formulations, there is a growing interest in the development of solid protein formulations due to improved protein stability in the solid state. We used small-scale (<3 g) ram and twin-screw extrusion for the solid stabilization of proteins (Lysozyme, BSA, and human insulin) in PEG-matrices. Protein stability after extrusion was systematically investigated using ss-DSC, ss-FTIR, CD spectroscopy, SEM-EDX, SEC, RP-HPLC, and in case of Lysozyme an activity assay. The applied analytical methods offered an accurate assessment of protein stability in extrudates, enabling the comparison of different melt extrusion formulations and process parameters (e.g., shear stress levels, screw configurations, residence times). Lysozyme was implemented as a model protein and was completely recovered in its active form after extrusion. Differences seen between Lysozyme- and BSA- or human insulin-loaded extrudates indicated that melt extrusion could have an impact on the conformational stability. In particular, BSA and human insulin were more susceptible to heat exposure and shear stress compared to Lysozyme, where shear stress was the dominant parameter. Consequently, ram extrusion led to less conformational changes compared to TSE. Ram extrusion showed good protein particle distribution resulting in the preferred method to prepare highly-loaded solid protein formulations.

Identification of neutral genome integration sites with high expression and high integration efficiency in Fusarium venenatum TB01.

CRISPR/Cas9-mediated homology-directed recombination is an efficient method to express target genes. Based on the above method, providing ideal neutral integration sites can ensure the reliable, stable, and high expression of target genes. In this study, we obtained a fluorescent transformant with neutral integration and high expression of the GFP expression cassette from the constructed GFP expression library and named strain FS. The integration site mapped at 4886 bp upstream of the gene FVRRES_00686 was identified in strain FS based on a Y-shaped adaptor-dependent extension, and the sequence containing 600 bp upstream and downstream of this site was selected as the candidate region for designing sgRNAs (Sites) for CRISPR/Cas9-mediated homology-directed recombination. PCR analysis showed that the integration efficiency of CRISPR/Cas9-mediated integration of target genes in designed sites reached 100%. Further expression stability and applicability analysis revealed that the integration of the target gene into the above designed sites can be stably inherited and expressed and has no negative effect on the growth of F. venenatum TB01. These results indicate the above designed neutral sites have the potential to accelerate the development of F. venenatum TB01 through overexpression of target genes in metabolic engineering.

Extracellular vesicle-loaded hydrogels for tissue repair and regeneration.

Extracellular vesicles (EVs) are a collective term for nanoscale or microscale vesicles secreted by cells that play important biological roles. Mesenchymal stem cells are a class of cells with the potential for self-healing and multidirectional differentiation. In recent years, numerous studies have shown that EVs, especially those secreted by mesenchymal stem cells, can promote the repair and regeneration of various tissues and, thus, have significant potential in regenerative medicine. However, due to the rapid clearance capacity of the circulatory system, EVs are barely able to act persistently at specific sites for repair of target tissues. Hydrogels have good biocompatibility and loose and porous structural properties that allow them to serve as EV carriers, thereby prolonging the retention in certain specific areas and slowing the release of EVs. When EVs are needed to function at specific sites, the EV-loaded hydrogels can stand as an excellent approach. In this review, we first introduce the sources, roles, and extraction and characterization methods of EVs and describe their current application status. We then review the different types of hydrogels and discuss factors influencing their abilities to carry and release EVs. We summarize several strategies for loading EVs into hydrogels and characterizing EV-loaded hydrogels. Furthermore, we discuss application strategies for EV-loaded hydrogels and review their specific applications in tissue regeneration and repair. This article concludes with a summary of the current state of research on EV-loaded hydrogels and an outlook on future research directions, which we hope will provide promising ideas for researchers.

The conundrum of COVID-19 mRNA vaccine-induced anaphylaxis.

Novel messenger RNA (mRNA) vaccines have proven to be effective tools against coronavirus disease 2019, and they have changed the course of the pandemic. However, early reports of mRNA vaccine-induced anaphylaxis resulted in public alarm, contributing toward vaccine hesitancy. Although initial reports were concerning for an unusually high rate of anaphylaxis to the mRNA vaccines, the true incidence is likely comparable with other vaccines. These reactions occurred predominantly in young to middle-aged females, and many had a history of allergies. Although initially thought to be triggered by polyethylene glycol (PEG), lack of reproducibility of these reactions with subsequent dosing and absent PEG sensitization point away from an IgE-mediated PEG allergy in most. PEG skin testing has poor posttest probability and should be reserved for evaluating non-vaccine-related PEG allergy without influencing decisions for subsequent mRNA vaccination. Immunization stress-related response can closely mimic vaccine-induced anaphylaxis and warrants consideration as a potential etiology. Current evidence suggests that many individuals who developed anaphylaxis to the first dose of an mRNA vaccine can likely receive a subsequent dose after careful evaluation. The need to understand these reactions mechanistically remains critical because the mRNA platform is rapidly finding its way into other vaccinations and therapeutics.

PEG allergy - A COVID-19 pandemic-made problem? A German perspective.

Background: Polyethylene glycol (PEG) has been used for decades, but only caused allergic reactions exceptionally. Introduction of PEG-containing COVID-19 vaccines might have fostered public interest beyond medical reasoning. Objectives: To investigate the impact of the SARS-CoV-2 pandemic on the public interest in PEG allergy in Germany and the published PEG allergy cases worldwide. Methods: A retrospective longitudinal study was conducted to measure public interest in PEG allergy analyzing Google search volume in Germany from February 2018 to January 2022. Medically confirmed "PEG allergy" cases were analyzed by looking at the numbers of PubMed case reports and case series from 1977 until January 2022. Results: Web results in Germany before COVID-19 show search volumes related to "PEG allergy/testing" was negligible, with 10 search queries per month. The pandemic led to a >200-fold increase from 250 queries 2 years before to 55 720 queries 2 years thereafter, reflecting tremendous public interest. Additionally, the maximum monthly search volume from before to during the pandemic increased immensely for "vaccination" (57-fold), "vaccination and adverse effects" (85-fold), "vaccination and allergy" (71-fold). In contrast, the increase of publication numbers for the search term "PEG allergy" was small from 2019 to 2021 (2.5-fold). Only a very low number of 211 cases with "PEG allergy" worldwide since 1977 could be identified. Conclusion: PEG allergy became a topic of major public interest because of COVID-19 vaccination. Scientific publications have increased to a lesser extent, probably promoted by public awareness. Conversely, the overall number of cases published with PEG allergy remain very low. The current high demand for COVID-19 vaccination allergy testing is triggered by public interest instead of medical reasoning.

Second-dose mRNA COVID-19 vaccine safety in patients with immediate reactions after the first dose: A case series.

Background: The rates of suspected allergic reactions to the first dose of the coronavirus disease 2019 (COVID-19) mRNA vaccines have been reported to be as high as 2%, with an anaphylaxis incidence up to 2.5 per 10,000 individuals. Anaphylaxis in response to the first dose may be considered a contraindication to administration of the second dose, even though the second dose is necessary for optimal protection against severe disease. Many individuals with anaphylactic reactions to the first dose still want to receive a second dose. However, there are few published data to support the safety of administration of a second dose in this population. Objective: The primary objective of this study was to determine the percentage of patients tolerating a second COVID-19 mRNA vaccine dose after an immediate reaction to the first dose. Methods: This was a retrospective chart review of 47 patients at a Canadian hospital who had immediate, suspected allergic reactions following their first COVID-19 mRNA vaccine dose and received a second dose within our allergy clinic. Results: Of 47 patients, 46 tolerated the second dose; 43% of patients developed mild, transient symptoms. There were no patients who developed anaphylaxis or needed epinephrine after the second dose. Conclusion: Our case series adds to current evidence that administration of a second COVID-19 mRNA vaccine dose has a good safety profile in patients with a history of immediate reactions after the first dose, including those with a history of anaphylaxis in response to the first dose.

Nanoparticles design considerations to co-deliver nucleic acids and anti-cancer drugs for chemoresistance reversal.

Chemoresistance and hence the consequent treatment failure is considerably challenging in clinical cancer therapeutics. The understanding of the genetic variations in chemoresistance acquisition encouraged the use of gene modulatory approaches to restore anti-cancer drug efficacy. Many smart nanoparticles are designed and optimized to mediate combinational therapy between nucleic acid and anti-cancer drugs. This review aims to define a rational design of such co-loaded nanocarriers with the aim of chemoresistance reversal at various cellular levels to improve the therapeutic outcome of anticancer treatment. Going through the principles of therapeutics loading, physicochemical characteristics tuning, and different nanocarrier modifications, also looking at combination effectiveness on chemosensitivity restoration. Up to now, these emerging nanocarriers are in development status but are expected to introduce outstanding outcomes.

Vaccination counseling with and without excipient skin testing in patients with suspected allergic reactions to mRNA COVID-19 vaccines and patients with atopy.

Background: Allergic reactions have been reported with mRNA vaccines for COVID-19 prevention. Patients perceived to be at higher risk for a reaction may be referred to an allergist, although evaluation strategies may differ between allergists. Objective: Our aim was to determine outcomes of COVID-19 vaccinations in patients evaluated by an allergist using different approaches. Methods: We conducted a retrospective case series evaluation of 98 patients seen at the University of Michigan Allergy Clinic for concerns regarding COVID-19 vaccination. Of these 98 patients, 34 underwent skin testing with polyethylene glycol (PEG) 2000 with or without PEG 3350/polysorbate 80 testing. Results: Of the 34 patients on whom skin testing was performed, 16 underwent testing before vaccination and 18 underwent testing after a reported vaccine-related event. One patient had a positive skin testing result in response to PEG 3350 following a vaccination reaction and natural infection and was advised against a second dose. One patient with a significant history concerning of anaphylaxis in response to PEG had positive results of testing to identify allergy to PEG 2000, PEG 3350, and polysorbate 80 and was advised against vaccination. Of the 98 patients, 63 (64%) tolerated COVID-19 vaccination without complication after evaluation by an allergist. Conclusion: No significant differences were found between vaccination counseling with and without skin testing to excipients. Patients who presented before the first dose of vaccination were more likely to proceed with COVID-19 vaccination and tolerate vaccination without complication.

Safety and efficacy of graded dosing of Pfizer-BioNTech mRNA COVID-19 vaccine after an immediate hypersensitivity reaction to first dose.

Current guidelines do not recommend subsequent mRNA COVID-19 vaccination in patients who experience immediate allergic reactions to the first dose. Our findings indicate that graded dosing of this vaccine is safe, efficacious, and useful for treating these individuals with allergy.

Roles and current applications of S-nitrosoglutathione in anti-infective biomaterials.

Bacterial infections can compromise the physical and biological functionalities of humans and pose a huge economical and psychological burden on infected patients. Nitric oxide (NO) is a broad-spectrum antimicrobial agent, whose mechanism of action is not affected by bacterial resistance. S-nitrosoglutathione (GSNO), an endogenous donor and carrier of NO, has gained increasing attention because of its potent antibacterial activity and efficient biocompatibility. Significant breakthroughs have been made in the application of GSNO in biomaterials. This review is based on the existing evidence that comprehensively summarizes the progress of antimicrobial GSNO applications focusing on their anti-infective performance, underlying antibacterial mechanisms, and application in anti-infective biomaterials. We provide an accurate overview of the roles and applications of GSNO in antibacterial biomaterials and shed new light on the avenues for future studies.

Single domain antibodies derived from ancient animals as broadly neutralizing agents for SARS-CoV-2 and other coronaviruses.

With severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as an emergent human virus since December 2019, the world population is susceptible to coronavirus disease 2019 (COVID-19). SARS-CoV-2 has higher transmissibility than the previous coronaviruses, associated by the ribonucleic acid (RNA) virus nature with high mutation rate, caused SARS-CoV-2 variants to arise while circulating worldwide. Neutralizing antibodies are identified as immediate and direct-acting therapeutic against COVID-19. Single-domain antibodies (sdAbs), as small biomolecules with non-complex structure and intrinsic stability, can acquire antigen-binding capabilities comparable to conventional antibodies, which serve as an attractive neutralizing solution. SARS-CoV-2 spike protein attaches to human angiotensin-converting enzyme 2 (ACE2) receptor on lung epithelial cells to initiate viral infection, serves as potential therapeutic target. sdAbs have shown broad neutralization towards SARS-CoV-2 with various mutations, effectively stop and prevent infection while efficiently block mutational escape. In addition, sdAbs can be developed into multivalent antibodies or inhaled biotherapeutics against COVID-19.

Biochemical, structural, and computational studies of a γ-carbonic anhydrase from the pathogenic bacterium Burkholderia pseudomallei.

Melioidosis is a severe disease caused by the highly pathogenic gram-negative bacterium Burkholderia pseudomallei. Several studies have highlighted the broad resistance of this pathogen to many antibiotics and pointed out the pivotal importance of improving the pharmacological arsenal against it. Since γ-carbonic anhydrases (γ-CAs) have been recently introduced as potential and novel antibacterial drug targets, in this paper, we report a detailed characterization of BpsγCA, a γ-CA from B. pseudomallei by a multidisciplinary approach. In particular, the enzyme was recombinantly produced and biochemically characterized. Its catalytic activity at different pH values was measured, the crystal structure was determined and theoretical pKa calculations were carried out. Results provided a snapshot of the enzyme active site and dissected the role of residues involved in the catalytic mechanism and ligand recognition. These findings are an important starting point for developing new anti-melioidosis drugs targeting BpsγCA.

Comparison of two chromogranin A assays and investigation of nonlinear specimens.

Background: As a marker for functional and non-functional neuroendocrine tumors, serum chromogranin A (CgA) concentrations have shown value for detecting and monitoring disease. Here we describe a comparison between an established micro-titer plate assay (Cisbio CgA ELISA) and an analyzer-based assay (B·R·A·H·M·S CgA II KRYPTOR). Reference limits were established along with a performance evaluation of the KRYPTOR assay. Nonlinearity observed in approximately 0.03% of patients was also investigated. Methods: Samples were tested according to kit manufacturer's protocols. Reference limits were established for both assays testing the same cohort of healthy volunteers. Potential causes of nonlinearity investigated were HAMA, macromolecule effects and elevated serum creatinine. Results: KRYPTOR vs. Cisbio: slope=0.692, y-intercept=-40.0 (r2=0.967, n=186). Upper reference limits were 160 and 103 ng/mL for the Cisbio and KRYPTOR assays, respectively. Linearity: slope=1.012 (r2=0.998) with 95.0-105.5% recoveries. Precision: repeatability ≤2.4%, within-laboratory ≤3.1% (79 and 738 ng/mL). Limit of detection: 8 ng/mL. Strong nonlinear specimens (n=6) retested for HAMA interference generated differences (block-no block) ranging -3.2-4.2%. Polyethylene glycol precipitation recoveries ranged from 157 to >5714% for affected specimens versus 71-79% for normal specimens. Eight of 14 nonlinear specimens (57%) had elevated serum creatinine results (>1.20 mg/dL). Conclusions: The CgA II KRYPTOR assay performs acceptably for quantifying CgA in human serum. While adequate correlation is observed against the Cisbio ELISA, there is significant disagreement overall. Efforts to identify a cause of the nonlinearity observed in a small percentage of patients were inconclusive, but neither HAMA interference, macromolecule effects nor renal failure appear as major factors.

Novel Drugs for the Management of Hepatic Encephalopathy: Still a Long Journey to Travel.

Hepatic encephalopathy (HE) is one of the reversible complications of chronic liver disease, associated with a higher mortality rate. In current clinical practice, treatment with rifaximin and lactulose/lactitol is the first line of treatment in HE. With the advance in pathophysiology, a new class of ammonia lowering drugs has been revealed to overcome the hurdle and disease burden. The mechanism of the novel agents differs significantly and includes the alteration in intestinal microbiota, intestinal endothelial integrity, oxidative stress, inflammatory markers, and modulation of neurotoxins. Most of the trials have reported promising results in the treatment and prevention of HE with fecal microbiota transplantation, albumin, probiotics, flumazenil, polyethylene glycol, AST-120, glycerol phenylbutyrate, nitazoxanide, branched-chain amino acid, naloxone, and acetyl-l-carnitine. However, their clinical use is limited due to the presence of major drawbacks in their study design, sample size, safety profile, bias, and heterogenicity. This study will discuss the novel therapeutic targets for HE in liver cirrhosis patients with supporting clinical trial data.

DNA sliding and loop formation by E. coli SMC complex: MukBEF.

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.