Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.

OBJECTIVE: Data on the safety and effectiveness of tenofovir alafenamide (TAF) plus peginterferon-alpha (Peg-IFN-α) in children with chronic hepatitis B (CHB) are lacking. The current study aimed to present the characteristics of four pediatric CHB patients who obtained a functional cure by using TAF and Peg-IFN-α.
METHODS: In this case series study initiated in May 2019, ten children who had no clinical symptoms or signs received response-guided (HBV DNA undetectable, hepatitis B e antigen [HBeAg] loss or seroconversion, and hepatitis B surface antigen [HBsAg] loss or seroconversion) and functional cure-targeted (HBsAg loss or seroconversion) TAF (25 mg/d, orally) plus Peg-IFN-α-2b (180 µg/1.73m2, subcutaneously, once weekly) in combination (9/10) or sequential (1/10) therapy. The safety and effectiveness of these treatments were monitored.
RESULTS: As of April 2024, four out of ten children obtained a functional cure after a mean of 31.5 months of treatment, and the other six children are still undergoing treatment. These four cured children, aged 2, 4, 8, and 6 years, were all HBeAg-positive and had alanine aminotransferase levels of 80, 47, 114, and 40 U/L; HBV DNA levels of 71200000, 93000000, 8220, and 96700000 IU/mL; and HBsAg levels of 39442.8, 15431.2, 22, and 33013.1 IU/mL, respectively. During treatment, all the children (10/10) experienced mild or moderate adverse events, including flu-like symptoms, anorexia, fatigue, and cytopenia. Notably, growth retardation (8/10) was the most significant adverse event; and it occurred in three cured children (3/4) treated with combination therapy and was present to a low degree in the other cured child (1/4) treated with sequential therapy. Fortunately, all three cured children recovered to or exceeded the normal growth levels at 9 months posttreatment.
CONCLUSIONS: TAF plus Peg-IFN-α-2b therapy is potentially safe and effective for pediatric CHB patients, which may provide important insights for future clinical practice and study designs targeting functional cures for children with CHB.

BACKGROUND: A trial performed among unvaccinated, high-risk outpatients with COVID-19 during the delta period showed remdesivir reduced hospitalization. We used our real-world data platform to determine the effectiveness of remdesivir on reducing 28-day hospitalization among outpatients with mild-moderate COVID-19 during an Omicron period including BQ.1/BQ.1.1/XBB.1.5.
METHODS: We did a propensity-matched, retrospective cohort study of non-hospitalized adults with SARS-CoV-2 infection between April 7, 2022, and February 7, 2023. Electronic healthcare record data from a large health system in Colorado were linked to statewide vaccination and mortality data. We included patients with a positive SARS-CoV-2 test or outpatient remdesivir administration. Exclusion criteria were other SARS-CoV-2 treatments or positive SARS-CoV-2 test more than seven days before remdesivir. The primary outcome was all-cause hospitalization up to day 28. Secondary outcomes included 28-day COVID-related hospitalization and 28-day all-cause mortality.
RESULTS: Among 29,270 patients with SARS-CoV-2 infection, 1,252 remdesivir-treated patients were matched to 2,499 untreated patients. Remdesivir was associated with lower 28-day all-cause hospitalization (1.3% vs. 3.3%, adjusted hazard ratio (aHR) 0.39 [95% CI 0.23-0.67], p < 0.001) than no treatment. All-cause mortality at 28 days was numerically lower among remdesivir-treated patients (0.1% vs. 0.4%; aOR 0.32 [95% CI 0.03-1.40]). Similar benefit of RDV treatment on 28-day all-cause hospitalization was observed across Omicron periods, aOR (95% CI): BA.2/BA2.12.1 (0.77[0.19-2.41]), BA.4/5 (0.50[95% CI 0.50-1.01]), BQ.1/BQ.1.1/XBB.1.5 (0.21[95% CI 0.08-0.57].
CONCLUSIONS: Among outpatients with SARS-CoV-2 during recent Omicron surges, remdesivir was associated with lower hospitalization than no treatment, supporting current National Institutes of Health Guidelines.

The formation pathway and mechanism of various pyrazines were investigated during the thermal treatment of the alanine-xylose Amadori compound (Ala-ARP) and exogenous alanine (Ala). 15N-labeled Ala was used to coheated with Ala-ARP to clarify the nitrogen sources and the respective contributions of exogenous Ala and the regenerated Ala released from Ala-ARP to different pyrazine formation. It was found that exogenous Ala exhibited a priority in capturing glyoxal (GO) to form pyrazine during the thermal degradation of ARP. Compared to the Ala-methylglyoxal (MGO) model, a lower activation energy was required for the Ala-GO reaction, where the reaction dynamics of Ala-GO followed a zero-order model. In addition to forming pyrazine, the interaction between existing exogenous Ala and GO would accelerate the thermal degradation of Ala-ARP and retro-aldolization reaction of deoxyxylosones (DXs) to α-dicarbonyls. During this process, the release of regenerated Ala and MGO was promoted. Accordingly, as GO was expended by exogenous Ala during the initial stage of ARP-Ala degradation, the condensation between regenerated Ala and MGO became intensified, leading to the generation of methylpyrazine and 2,5-dimethylpyrazine. As a result, in the thermally treated mixture of Ala-ARP and exogenous Ala, 55% of the formed pyrazine originated from exogenous Ala, while 63% of the formed methylpyrazine and 57% of the formed 2,5-dimethylpyrazine were derived from regenerated Ala (120 °C, 30 min).

Brain rhythms provide the timing for recruitment of brain activity required for linking together neuronal ensembles engaged in specific tasks. The γ-oscillations (30 to 120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimer\'s disease (AD). Here, we report on a potent brain-permeable small molecule, DDL-920 that increases γ-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a class of therapeutics for AD. We employed anatomical, in vitro and in vivo electrophysiological, and behavioral methods to examine the effects of our lead therapeutic candidate small molecule. As a novel in central nervous system pharmacotherapy, our lead molecule acts as a potent, efficacious, and selective negative allosteric modulator of the γ-aminobutyric acid type A receptors most likely assembled from α1β2δ subunits. These receptors, identified through anatomical and pharmacological means, underlie the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of γ-oscillations. When orally administered twice daily for 2 wk, DDL-920 restored the cognitive/memory impairments of 3- to 4-mo-old AD model mice as measured by their performance in the Barnes maze. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brain\'s endogenous γ-oscillations through enhancing the function of PV+INs.

The dynamics of lysozyme is probed by attaching -SCN to all alanine residues. The one-dimensional infrared spectra exhibit frequency shifts in the position of the maximum absorption of 4 cm-1, which is consistent with experiments in different solvents and indicates moderately strong interactions of the vibrational probe with its environment. Isotopic substitution 12C → 13C leads to a redshift by -47 cm-1, which agrees quantitatively with experiments for CN-substituted copper complexes in solution. The low-frequency, far-infrared part of the protein spectra contains label-specific information in the difference spectra when compared with the wild type protein. Depending on the position of the labels, local structural changes are observed. For example, introducing the -SCN label at Ala129 leads to breaking of the α-helical structure with concomitant change in the far-infrared spectrum. Finally, changes in the local hydration of SCN-labeled alanine residues as a function of time can be related to the reorientation of the label. It is concluded that -SCN is potentially useful for probing protein dynamics, both in the high-frequency part (CN-stretch) and in the far-infrared part of the spectrum.

This letter to the editor relates to the study entitled \"Tenofovir amibufenamide vs tenofovir alafenamide for treating chronic hepatitis B: A real-world study\", which was recently published by Peng et al. Hepatitis B virus infection represents a significant health burden worldwide and can lead to cirrhosis and even liver cancer. The antiviral drugs currently used to treat patients with chronic hepatitis B infection still have many side effects, so it is crucial to identify safe and effective drugs to inhibit viral replication.

This study introduces a novel method named multiple parameter replica exchange Gaussian accelerated molecular dynamics (MP-Rex-GaMD), building on the Gaussian accelerated molecular dynamics (GaMD) algorithm. GaMD enhances sampling and retrieves free energy information for biomolecular systems by adding a harmonic boost potential to smooth the potential energy surface without the need for predefined reaction coordinates. Our innovative approach advances the acceleration power and energetic reweighting accuracy of GaMD by incorporating a replica exchange algorithm that enables the exchange of multiple parameters, including the GaMD boost parameters of force constant and energy threshold, as well as temperature. Applying MP-Rex-GaMD to the three model systems of dialanine, chignolin, and HIV protease, we demonstrate its superior capability over conventional molecular dynamics and GaMD simulations in exploring protein conformations and effectively navigating various biomolecular states across energy barriers. MP-Rex-GaMD allows users to accurately map free energy landscapes through energetic reweighting, capturing the ensemble of biomolecular states from low-energy conformations to rare high-energy transitions within practical computational time scales.

Chemical mutagenesis via dehydroalanine (Dha) is a powerful method to tailor protein structure and function, allowing the site-specific installation of post-translational modifications and non-natural functional groups. Despite the impressive versatility of this method, applications have been limited, as products are formed as epimeric mixtures, whereby the modified amino acid is present as both the desired l-configuration and a roughly equal amount of the undesired d-isomer. Here, we describe a simple remedy for this issue: removal of the d-isomer via proteolysis using a d-stereoselective peptidase, alkaline d-peptidase (AD-P). We demonstrate that AD-P can selectively cleave the d-isomer of epimeric residues within histone H3, GFP, Ddx4, and SGTA, allowing the installation of non-natural amino acids with stereochemical control. Given the breadth of modifications that can be introduced via Dha and the simplicity of our method, we believe that stereoselective chemoenzymatic mutagenesis will find broad utility in protein engineering and chemical biology applications.

Sepsis is characterized by a metabolic disorder of amino acid occurs in the early stage; however, the profile of serum amino acids and their alterations associated with the onset of sepsis remain unclear. Thus, our objective is to identify the specific kinds of amino acids as diagnostic biomarkers in pediatric patients with sepsis. Serum samples were collected from patients with sepsis admitted to the pediatric intensive care unit (PICU) between January 2019 and December 2019 on the 1st, 3rd and 7th day following admission. Demographic and laboratory variables were also retrieved from the medical records specified times. Serum amino acid concentrations were detected by UPLC-MS/MS system. PLS-DA (VIP > 1.0) and Kruskal-Wallis test (p < 0.05) were employed to identify potential biomarkers. Spearman\'s rank correlation analysis was conducted to find the potential association between amino acid levels and clinical features. The diagnostic utility for pediatric sepsis was assessed using receiver operating characteristic (ROC) curve analysis. Most of amino acid contents in serum were significantly decreased in patients with sepsis, but approached normal levels by the seventh day post-diagnosis. Threonine (THR), lysine (LYS), valine (VAL) and alanine (ALA) emerged as potential biomarkers related for sepsis occurrence, though they were not associated with PELOD/PELOD-2 scores. Moreover, alterations in serum THR, LYS and ALA were linked to complications of brain injury, and serum ALA levels were also related to sepsis-associated acute kidney injury. Further analysis revealed that ALA was significantly correlated with the Glasgow score, serum lactate and glucose levels, C-reactive protein (CRP), and other indicators for liver or kidney dysfunction. Notably, the area under the ROC curve (AUC) for ALA in distinguishing sepsis from healthy controls was 0.977 (95% CI: 0.925-1.000). The serum amino acid profile of children with sepsis is significantly altered compared to that of healthy controls. Notably, ALA shows promise as a potential biomarker for the early diagnosis in septic children.