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Up to one-third of individuals suffering from acute SARS-CoV-2 infection with the onset of severe-to-mild diseases could develop several symptoms of neurological disorders, which could last long after resolving the infection, known as neuro-COVID. Effective therapeutic treatments for neuro-COVID remain unavailable, in part, due to the absence of animal models for studying its underlying mechanisms and developing medical countermeasures against it. Here, we explored the impact of SARS-CoV-2 infection on the well-being of respiratory and neurological functions of BALB/c mice by using a clinical isolate of β-variant, i.e. B.1.351. We found that this β-variant of SARS-CoV-2 primarily infected the lungs, causing tissue damage, profound inflammatory responses, altered respiratory functions and transient but significant hypoxia. Although live progeny viruses could not be isolated, viral RNAs were detected across many anatomical regions of the brains in most challenged mice and triggered activation of genes encoding for NF -kB , IL-6 , IP-10 and RANTES and microglial cells. We noted that the significantly activated IL-6 -encoded gene persisted at 4 weeks after infection. Together, these results suggest that this B.1.351/BALB/c model of SARS-CoV-2 infection warrants further studies to establish it as a desirable model for studies of neuropathogenesis and the development of effective therapeutics of neuro-COVID.

Background: The prognosis for patients with esophageal cancer who received neoadjuvant chemoradiotherapy (nCRT) followed by surgery has shown improvement in recent years. We sought to identify the critical factors contributing to enhanced survival outcomes., Patients and Methods: We retrospectively examined 427 patients with esophageal cancer treated with nCRT and esophagectomy across two periods: P1 (from 1 January 2004 to 31 December 2011) and P2 (from 1 January 2012 to 31 December 2017). The introduction of the CROSS regimen and total meso-esophagectomy in P2 prompted an evaluation of their effects on perioperative outcomes and overall survival (OS)., Results: During P2, the occurrence of recurrent laryngeal nerve palsy increased significantly from 3.9 to 16.8% (p < 0.001), while pneumonia and in-hospital mortality rates remained unchanged. The median OS improved from 19.2 to 29.2 months (p < 0.001) between P1 and P2. Multivariable analysis identified higher nodal yields and the achievement of major response as favorable prognostic factors. Conversely, an involved circumferential resection margin (CRM), an advanced ypN stage, and pneumonia were independently associated with poor outcomes. Patients treated during P2 had a lower prevalence of involved CRM (10% vs. 25.1%, p < 0.001), a higher rate of major response (52.7% vs. 34.8%, p < 0.01), and a greater nodal yield (27.8 vs. 10.9, p < 0.001)., Conclusions: The clinical outcomes following nCRT and surgery have improved significantly over time. This progress can be attributed to multiple factors, with the primary drivers being the refinement of nCRT protocols and the application of radical surgery., (© 2024. Society of Surgical Oncology.)

COVID-19 presents with a plethora of neurological signs and symptoms despite being characterized as a respiratory disease, including seizures, anxiety, depression, amnesia, attention deficits, and alterations in consciousness. The olfactory nerve is widely accepted as the neuroinvasive route by which the etiological agent SARS-CoV-2 enters the brain, but the trigeminal nerve is an often-overlooked additional route. Based on this consensus, we initially conducted a pilot experiment investigating the olfactory nerve route of SARS-CoV-2 neuroinvasion via intranasal inoculation in AC70 human ACE2 transgenic mice. Notably, we found that the trigeminal ganglion is an early and highly efficient site of viral replication, which then rapidly spread widely throughout the brain where neurons were primarily targeted. Despite the extensive viral infection across the brain, obvious evidence of tissue pathology including inflammatory infiltration, glial activation, and apoptotic cell deaths were not consistently observed, albeit inflammatory cytokines were significantly induced. However, the expression levels of different genes related to neuronal function, including the neurotransmitter dopamine pathway as well as synaptic function, and markers of neuronal damage were altered as compared to mock-infected mice. Our findings suggest that the trigeminal nerve may serve as a neuroinvasive route complementary to the olfactory nerve and that the ensuing neuroinvasion presented a unique neuropathological profile. This study provides insights into potential neuropathogenic mechanisms utilized by coronaviruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hsu, Saenkham-Huntsinger, Huang, Octaviani, Drelich, Peng and Tseng.)

We refresh the design of zero-mode waveguides (ZMWs) by introducing metamaterials that makes the zeroth order resonant mode existence. Of particular importance, the resulting electromagnetic field exhibits nearly constant distribution but not a trivial solution of Maxwell's equation, showing great advantage to equalize the excitation rate of molecules throughout the waveguides. A closed form expression for the wave impedance is derived which is verified by the finite-difference time-domain simulations. Benefitted from the cavity Purcell effect which is lacking in existing ZMWs, fluorescence amplification and lifetime reduction are simultaneously enhanced. A practical design where the excitation volume reduced down to sub-zeptoliter and the fluorescence lifetime shortened to picosecond scale is illustrated. This result makes single molecule real time (SMRT) sensing of biochemical reactions at biophysically relevant concentrations (~ μM) possible, combining off-the-shelf ultrafast lasers., (© 2024. The Author(s).)

Human telomerase assembly is a highly dynamic process. Using biochemical approaches, we find that LARP3 and LARP7/MePCE are involved in the early stage of human telomerase RNA (hTR) and that their binding to RNA is destabilized when the mature form is produced. LARP3 plays a negative role in preventing the processing of the 3'-extended long (exL) form and the binding of LARP7 and MePCE. Interestingly, the tertiary structure of the exL form prevents LARP3 binding and facilitates hTR biogenesis. Furthermore, low levels of LARP3 promote hTR maturation, increase telomerase activity, and elongate telomeres. LARP7 and MePCE depletion inhibits the conversion of the 3'-extended short (exS) form into mature hTR and the cytoplasmic accumulation of hTR, resulting in telomere shortening. Taken together our data suggest that LARP3 and LARP7/MePCE mediate the processing of hTR precursors and regulate the production of functional telomerase., (© 2024. The Author(s).)

Infiltrative non-GBM gliomas are common primary intracranial malignancies, and postoperative adjuvant radiotherapy is recommended for most adult patients diagnosed with this disease to enhance local control and prolong intracranial progression-free survival (PFS). However, RT-related neurocognitive function (NCF) consequences should not be ignored. Early neurocognitive decline principally includes episodic memory, associated significantly with functions of the hippocampus. This prospective study aims to investigate the impact of adjuvant brain irradiation on neurocognitive performances and relevant oncological outcomes.Twenty-five patients with intracranial infiltrative non-GBM gliomas were enrolled when postoperative adjuvant RT was recommended. All recruited patients should receive baseline brain magnetic resonance imaging, and neuropsychological assessments before and 4 months after the RT course. A battery of neuropsychological measures, mainly including executive functions, memory, psychomotor speed and visuoconstructive ability, was used to evaluate NCFs of interest.Analyzing the delta values between post-irradiation and baseline NCF scores, we observed a robust trend reflecting cognitive stabilization rather than deterioration in almost all NCF. Both verbal and visual memory functions exhibited significant differences in the corresponding scaled scores ( Z  = -2.722, p = .006, regarding verbal memory; Z  = -2.246, p = .025, concerning non-verbal memory). Moreover, patients' neuropsychological performances associated with psychomotor speed and executive functions also disclosed a tendency toward stabilization/improvement.This prospective study demonstrated that patients with infiltrative non-GBM exhibited a marked tendency toward neurocognitive stabilization after receiving postoperative adjuvant RT. Clinical trial registration: Trial Registration with ClinicalTrials.gov identifier: NCT03534050.

Coronavirus disease 2019 (COVID-19) continues to cause morbidity and mortality worldwide; therefore, effective treatments remain crucial to controlling it. As interferon-alpha (IFN-α) and -beta (β) have been proposed as COVID-19 treatments, we sought to assess their effectiveness on respiratory, cardiovascular, neurological, and psychiatric signs and symptoms, as well as PASC and death, in hospitalized COVID-19 patients without multiple sclerosis (MS). Using a federated data research network (TriNetX), we performed a retrospective cohort study of hospitalized COVID-19 patients without MS who received IFN-α or -β treatment, comparing them to a similar cohort who did not receive treatment. Following propensity-score matched analyses, we demonstrate that hospitalized COVID-19 patients who were treated with IFN-α or -β had significantly higher odds of death. In contrast, there was no significant difference in any other outcomes between 1-30 days or 1 day to anytime afterward. Overall, hospitalized COVID-19 patients without MS who were treated with IFN-α or -β had similar short- and long-term sequelae (except for mortality) as those who did not receive treatment. The potential benefits of utilizing IFN-α or -β treatment as therapeutics remain to be realized, and our research highlights the need to explore repurposing drugs for COVID-19 using real-world evidence.

COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS- CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly understood, largely due to the lack of suitable animal models that recapitulate key clinical and pathological symptoms. Here, we fully characterized AC70 line of human ACE2 transgenic (AC70 hACE2 Tg) mice for SARS-CoV-2 infection. We noted that this model is highly permissive to SARS-CoV-2 with values of 50% lethal dose and infectious dose as ~ 3 and ~ 0.5 TCID50 of SARS-CoV-2, respectively. Mice infected with 105 TCID50 of SARS-CoV-2 rapidly succumbed to infection with 100% mortality within 5 days. Lung and brain were the prime tissues harboring high viral titers, accompanied by histopathology. However, viral RNA and inflammatory mediators could be detectable in other organs, suggesting the nature of a systemic infection. Lethal challenge of AC70 hACE2 Tg mice caused acute onset of leukopenia, lymphopenia, along with an increased neutrophil-to-lymphocyte ratio (NLR). Importantly, infected animals recapitulated key features of COVID-19-associated coagulopathy. SARS-CoV-2 could induce the release of circulating neutrophil extracellular traps (NETs), along with activated platelet/endothelium marker. Immunohistochemical staining with anti-platelet factor-4 (PF4) antibody revealed profound platelet aggregates especially within blocked veins of the lungs. We showed that acute SARS-CoV-2 infection triggered a hypercoagulable state coexisting with ill-regulated fibrinolysis. Finally, we highlighted the potential role of Annexin A2 (ANXA2) in fibrinolytic failure. ANXA2 is a calcium-dependent phospholipid-binding protein that forms a heterotertrameric complexes localized at the extracellular membranes with two S100A10 small molecules acting as a co-receptor for tissue-plasminogen activator (t-PA), tightly involved in cell surface fibrinolysis. Thus, our results revealing elevated IgG type anti-ANXA2 antibody production, downregulated de novo ANXA2/S100A10 synthesis, and reduced ANXA2/S100A10 association in infected mice, this protein might serve as druggable targets for development of antithrombotic and/or anti-fibrinolytic agents to attenuate pathogenesis of COVID-19., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Drelich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) remains a public health concern and a subject of active research effort. Development of pre-clinical animal models is critical to study viral-host interaction, tissue tropism, disease mechanisms, therapeutic approaches, and long-term sequelae of infection. Here, we report two mouse models for studying SARS-CoV-2: A knock-in mAce2 F83Y,H353K mouse that expresses a mouse-human hybrid form of the angiotensin-converting enzyme 2 (ACE2) receptor under the endogenous mouse Ace2 promoter, and a Rosa26 conditional knock-in mouse carrying the human ACE2 allele (Rosa26 hACE2 ). Although the mAce2 F83Y,H353K mice were susceptible to intranasal inoculation with SARS-CoV-2, they did not show gross phenotypic abnormalities. Next, we generated a Rosa26 hACE2 ;CMV-Cre mouse line that ubiquitously expresses the human ACE2 receptor. By day 3 post infection with SARS-CoV-2, Rosa26 hACE2 ;CMV-Cre mice showed significant weight loss, a variable degree of alveolar wall thickening and reduced survival rates. Viral load measurements confirmed inoculation in lung and brain tissues of infected Rosa26 hACE2 ;CMV-Cre mice. The phenotypic spectrum displayed by our different mouse models translates to the broad range of clinical symptoms seen in the human patients and can serve as a resource for the community to model and explore both treatment strategies and long-term consequences of SARS-CoV-2 infection., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Recently, more and more total pancreatectomy (TP) has been performed for central-located pancreatic ductal cell adenocarcinoma (PDCA) which abuts or involves both gastroduodenal and splenic arteries and demands transaction of both of them for a complete resection. Spiked by Warshaw's procedure (spleen-preserving distal pancreatectomy with excision of splenic vessels), we developed a new procedure "Whipple over the splenic artery (WOTSA)" to replace TP by leftward extension of pancreatic parenchyma transaction line and preservation of pancreatic tail and spleen after excision of splenic artery. This uncontrolled before and after study assesses the safety and efficacy of a new technique "Whipple over the splenic artery (WOTSA)" as a treatment for PDAC which traditionally requires total pancreatectomy (TP) for a complete excision., Methods: The study group comprised 40 consecutive patients who underwent WOTSA for PDAC between August 2019 and September 2022. Their clinicopathological characteristics and survival were compared with those of a historical control group comprising 30 consecutive patients who underwent TP between January 2016 and July 2019., Results: None of the 40 patients in the WOTSA group required reoperation due to infarction of the pancreas and/or spleen remnant. DM medication after WOTSA were none in 19, oral hypoglycemic agents in 19, and insulin preparations in 2 patients. Compared with TP, patients who underwent WOTSA exhibited similar rates of major operative complications, clear pancreatic parenchyma transaction margin, and number of harvested positive lymph nodes, but higher rate of adjuvant chemotherapy completion and a trend toward better median disease free survival (14 vs. 7.5 mo, P=0.023)., Conclusions: Compared to TP, WOTSA can be safely performed and have much better postoperative glycemic status without cost of higher operative risk or impaired surgical radicality. These findings indicate that most TPs for PDAC potentially can be replaced by WOTSAs., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M pro ) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 M pro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

Introduction: Blood-brain barrier (BBB) remains to be the major obstacle to conquer in treating patients with malignant brain tumors. Radiation therapy (RT), despite being the mainstay adjuvant modality regardless of BBB, the effect of radiation induced cell death is hindered by the hypoxic microenvironment. Focused ultrasound (FUS) combined with systemic microbubbles has been shown not only to open BBB but also potentially increased regional perfusion. However, no clinical study has investigated the combination of RT with FUS-BBB opening (RT-FUS)., Methods: We aimed to provide preclinical evidence of RT-FUS combination in GBM animal model, and to report an interim analysis of an ongoing single arm, prospective, pilot study (NCT01628406) of combining RT-FUS for recurrent malignant high grade glioma patients, of whom re-RT was considered for disease control. In both preclinical and clinical studies, FUS-BBB opening was conducted within 2 h before RT. Treatment responses were evaluated by objective response rate (ORR) using magnetic resonance imaging, progression free survival, and overall survival, and adverse events (AE) in clinical study. Survival analysis was performed in preclinical study and descriptive analysis was performed in clinical study., Results: In mouse GBM model, the survival analysis showed RT-FUS (2 Gy) group was significantly longer than RT (2 Gy) group and control, but not RT (5 Gy) group. In the pilot clinical trial, an interim analysis of six recurrent malignant high grade glioma patients underwent a total of 24 RT-FUS treatments was presented. Three patients had rapid disease progression at a mean of 33 days after RT-FUS, while another three patients had at least stable disease (mean 323 days) after RT-FUS with or without salvage chemotherapy or target therapy. One patient had partial response after RT-FUS, making the ORR of 16.7%. There was no FUS-related AEs, but one (16.7%) re-RT-related grade three radiation necrosis., Conclusion: Reirradiation is becoming an option after disease recurrence for both primary and secondary malignant brain tumors since systemic therapy significantly prolongs survival in cancer patients. The mechanism behind the synergistic effect of RT-FUS in preclinical model needs further study. The clinical evidence from the interim analysis of an ongoing clinical trial (NCT01628406) showed a combination of RT-FUS was safe (no FUS-related adverse effect). A comprehensive analysis of radiation dosimetry and FUS energy distribution is expected after completing the final recruitment., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

For centuries, food, herbal medicines, and natural products have been valuable resources for discovering novel antiviral drugs, uncovering new structure-activity relationships, and developing effective strategies to prevent/treat viral infections. One such resource is Phellinus linteus, a mushroom used in folk medicine in Taiwan, Japan, Korea, and China. In this rich historical context, the key metabolites of Phellinus linteus mycelia ethanolic extract (GKPL) impacting the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at multiple stages have yet to be explored. Thus, this study systematically identifies and assesses the inhibitory effect of GKPL on the SARS-CoV-2 virus. Initially, the concentrations and contact times of GKPL against SARS-CoV-2 pseudovirus were assessed in HepG2 cells. Subsequently, utilizing the Ultra Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry method, potential biomarkers in the fungal extract were discerned. Metabolomic analysis identified 18 compounds in GKPL, with hispidin and hypholomine B present in the highest amounts. These compounds were isolated using chromatographic techniques and further identified through 1D NMR spectroscopic and mass spectrometry analysis. Hispidin and hypholomine B were found to inhibit the infection of SARS-CoV-2 pseudovirus by reducing angiotensin-converting enzyme 2 gene expression in HepG2, thereby decreasing viral entry. Moreover, hispidin and hypholomine B effectively block the spike receptor-binding domain, while hypholomine B, for the first time, showed significant inhibition of 3CL protease. This suggests that GKPL, enriched with hispidin and hypholomine B, has the potential to be used as an active ingredient against SARS-CoV-2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Licensed COVID-19 vaccines ameliorate viral infection by inducing production of neutralizing antibodies that bind the SARS-CoV-2 Spike protein and inhibit viral cellular entry. However, the clinical effectiveness of these vaccines is transitory as viral variants escape antibody neutralization. Effective vaccines that solely rely upon a T cell response to combat SARS-CoV-2 infection could be transformational because they can utilize highly conserved short pan-variant peptide epitopes, but a mRNA-LNP T cell vaccine has not been shown to provide effective anti-SARS-CoV-2 prophylaxis. Here we show a mRNA-LNP vaccine (MIT-T-COVID) based on highly conserved short peptide epitopes activates CD8 + and CD4 + T cell responses that attenuate morbidity and prevent mortality in HLA-A*02:01 transgenic mice infected with SARS-CoV-2 Beta (B.1.351). We found CD8 + T cells in mice immunized with MIT-T-COVID vaccine significantly increased from 1.1% to 24.0% of total pulmonary nucleated cells prior to and at 7 days post infection (dpi), respectively, indicating dynamic recruitment of circulating specific T cells into the infected lungs. Mice immunized with MIT-T-COVID had 2.8 (2 dpi) and 3.3 (7 dpi) times more lung infiltrating CD8 + T cells than unimmunized mice. Mice immunized with MIT-T-COVID had 17.4 times more lung infiltrating CD4 + T cells than unimmunized mice (7 dpi). The undetectable specific antibody response in MIT-T-COVID-immunized mice demonstrates specific T cell responses alone can effectively attenuate the pathogenesis of SARS-CoV-2 infection. Our results suggest further study is merited for pan-variant T cell vaccines, including for individuals that cannot produce neutralizing antibodies or to help mitigate Long COVID., Competing Interests: DG and BC are founders of Think Therapeutics. PL and YT are employees of Acuitas Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Carter, Huang, Liu, Liang, Lin, Peng, McKay, Dimitrakakis, Hsu, Tat, Saenkham-Huntsinger, Chen, Kaseke, Gaiha, Xu, Griffiths, Tam, Tseng and Gifford.)

As an important free energy source, the membrane voltage (V m ) regulates many essential physiological processes in bacteria. However, in comparison with eukaryotic cells, knowledge of bacterial electrophysiology is very limited. Here, we developed a set of novel genetically encoded bacterial V m sensors which allow single-cell recording of bacterial V m dynamics in live cells with high temporal resolution. Using these new sensors, we reveal the electrically "excitable" and "resting" states of bacterial cells dependent on their metabolic status. In the electrically excitable state, frequent hyperpolarization spikes in bacterial V m are observed, which are regulated by Na + /K + ratio of the medium and facilitate increased antibiotic tolerance. In the electrically resting state, bacterial V m displays significant cell-to-cell heterogeneity and is linked to the cell fate after antibiotic treatment. Our findings demonstrate the potential of our newly developed voltage sensors to reveal the underpinning connections between bacterial V m and antibiotic tolerance.

The spliceosome is a dynamic ribonucleoprotein particle and is assembled via sequential binding of five snRNAs and numerous protein factors. To understand the molecular mechanism of the splicing reaction, it is necessary to dissect the spliceosome pathway and isolate spliceosome intermediates in various stages of the pathway for biochemical and structural analysis. Here, we describe protocols for preparing intron-containing transcripts, cell-free splicing extracts, and in vitro splicing reactions, as well as procedures to arrest the spliceosome at different stages of the pathway for characterization of specific splicing complexes from the budding yeast Saccharomyces cerevisiae. Methods for arresting spliceosomes at specific stages include depletion with antibodies against factors required for specific steps of the pathway, use of extracts prepared from temperature-sensitive mutants, use of dominant negative mutants of DExD/H-box proteins, and use of mutant substrates., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacterial flagella are molecular machines used for motility and chemotaxis. The flagellum consists of a thin extracellular helical filament as a propeller, a short hook as a universal joint, and a basal body as a rotary motor. The filament is made up of more than 20,000 flagellin molecules and can grow to several micrometers long but only 20 nanometers thick. The regulation of flagellar assembly and ejection is important for bacterial environmental adaptation. However, due to the technical difficulty to observe these nanostructures in live cells, our understanding of the flagellar growth and loss is limited. In the last three decades, the development of fluorescence microscopy and fluorescence labeling of specific cellular structure has made it possible to perform the real-time observation of bacterial flagellar assembly and ejection processes. Furthermore, flagella are not only critical for bacterial motility but also important antigens stimulating host immune responses. The complete understanding of bacterial flagellar production and ejection is valuable for understanding macromolecular self-assembly, cell adaptation, and pathogen-host interactions., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The prognosis of patients with resected esophageal squamous cell carcinoma after neoadjuvant chemoradiotherapy is particularly poor in those who were staged as ypT3/T4 and/or ypN+. This study investigated whether adjuvant chemoradiotherapy was associated with improved clinical outcomes in these patients., Methods: we identified patients with esophageal squamous cell carcinoma who were staged as ypT3/T4 and/or ypN+ after being treated with neoadjuvant chemoradiotherapy followed by esophagectomy between the years 2013 and 2019. Patients were divided into two groups based on whether they received adjuvant chemoradiotherapy. The Kaplan-Meier method and Cox regression modeling were performed for survival analyses and multivariable analysis, respectively., Results: 76 eligible patients were included in the analyses. The median follow-up for the study cohort was 43.4 months. On Kaplan-Meier analyses of the overall population, adjuvant chemoradiotherapy was associated with significantly improved median overall survival (31.7 months vs. 16.3 months, p = 0.036). On Kaplan-Meier analyses of the 35 matched pairs generated by propensity score matching, adjuvant chemoradiotherapy was associated with significantly longer median overall survival (31.7 months vs. 14.3 months; p = 0.004) and median recurrence-free survival (18.9 months vs. 11.7 months; p = 0.020). In multivariable analysis, adjuvant chemoradiotherapy was independently associated with a 60% reduction in mortality ( p = 0.003) and a 48% reduction in risk of recurrence ( p = 0.035) after adjusting for putative confounders. In addition, microscopic positive resection margin and Mandard tumor regression grade 3-4 were independently associated with increased mortality and risk of recurrence. While a greater number of lymph nodes dissected was independently associated with significantly improved overall survival, the number of positive lymph nodes was independently associated with significantly worse overall survival and a trend ( p = 0.058) towards worse recurrence-free survival., Conclusions: This study demonstrated that adjuvant CRT was independently associated with a significantly improved survival and lower risk of recurrence than observation in esophageal squamous cell carcinoma patients staged as ypT3 and/or ypN+ after receiving neoadjuvant chemoradiotherapy and radical surgery. The results of this study have implications for the design of future clinical trials and may improve treatment outcomes of patients in this setting who cannot afford or are without access to adjuvant nivolumab.

Hoyeraal-Hreidarsson syndrome (HHS) is the most severe form of dyskeratosis congenita (DC) and is caused by mutations in genes involved in telomere maintenance. Here, we identified male siblings from a family with HHS carrying a hemizygous mutation (c.1345C > G, p.R449G), located in the C-terminal nuclear localization signal (NLS) of the DKC1 gene. These patients exhibit progressive cerebellar hypoplasia, recurrent infections, pancytopenia due to bone marrow failure, and short leukocyte telomere lengths. Single-cell RNA sequencing analysis suggested defects in the NLRP3 inflammasome in monocytes and the activation and maturation of NK cells and B cells. In experiments using induced pluripotent stem cells (iPSCs) from patients, DKC1&#95;R449G iPSCs had short telomere lengths due to reduced levels of human telomerase RNA (hTR) and increased cytosolic proportions of DKC1. Treatment with dihydroquinolizinone RG7834 and 3'deoxyanosine cordycepin rescued telomere length in patient-derived iPSCs. Together, our findings not only provide new insights into immunodeficiency in DC patients but also provide treatment options for telomerase insufficiency disorders., (© 2022. The Author(s).)

Background/aim: Neoadjuvant concurrent chemoradiotherapy (CCRT) for esophageal cancer is often overwhelming due to its toxic effects. This study aimed to establish a prognostic indicator based on pretreatment albumin and neutrophil-to-lymphocyte (NLR) ratio score (ANS) in comparison to the Prognostic Nutritional Index (PNI) in patients with esophageal cancer., Patients and Methods: A total of 123 patients who received neoadjuvant CCRT for esophageal cancer were prospectively and consecutively recruited between August 2016 and December 2017 from three medical institutes in Taiwan. Patients were assigned to ANS 0, 1, and 2 groups based on their pretreatment albumin and NLR values. ANS and PNI performances were compared for prediction of survival outcome., Results: Compared with ANS 0 (39 patients) and ANS 1 (51 patients), ANS 2 (33 patients) cases showed worse overall survival (hazard ratio=2.96; 95% confidence interval=1.45-6.05; log-rank p=0.003; hazard ratio=3.79; 95% confidence interval=1.79-8.02, p<0.001, respectively). ANS had better performance in overall survival evaluation and discrimination ability than PNI and individual albumin and NLR. Patients in the ANS 0, 1, and 2 had radiotherapy incompletion rates of 2.6%, 3.9%, and 18.2%, respectively, and chemotherapy incompletion rates of 5.1%, 7.8%, and 30.3%, respectively. Patients in the ANS 2 group were significantly associated with a higher incidence of infection (30.3%) than those in the ANS 0 (10.3%) and ANS 1 groups (9.8%)., Conclusion: Pre-treatment ANS was significantly associated with CCRT safety profiles, CCRT completion rate, and survival outcome in patients with esophageal cancer with excellent performance compared to PNI and NLR., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The U.S. Food and Drug Administration-authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected in two doses and effective in preventing COVID-19, but they do not induce efficient mucosal immunity or prevent viral transmission. Here, we report the first noninfectious, bacteriophage T4-based, multicomponent, needle- and adjuvant-free, mucosal vaccine harboring engineered Spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of two doses of this T4 SARS-CoV-2 vaccine 21 days apart induced robust mucosal immunity, in addition to strong systemic humoral and cellular immune responses. The intranasal vaccine induced broad virus neutralization antibody titers against multiple variants, Th1-biased cytokine responses, strong CD4 + and CD8 + T cell immunity, and high secretory IgA titers in sera and bronchoalveolar lavage specimens from vaccinated mice. All of these responses were much stronger in intranasally vaccinated mice than those induced by the injected vaccine. Furthermore, the nasal vaccine provided complete protection and sterilizing immunity against the mouse-adapted SARS-CoV-2 MA10 strain, the ancestral WA-1/2020 strain, and the most lethal Delta variant in both BALB/c and human angiotensin converting enzyme (hACE2) knock-in transgenic mouse models. In addition, the vaccine elicited virus-neutralizing antibodies against SARS-CoV-2 variants in bronchoalveolar lavage specimens, did not affect the gut microbiota, exhibited minimal lung lesions in vaccinated and challenged mice, and is completely stable at ambient temperature. This modular, needle-free, phage T4 mucosal vaccine delivery platform is therefore an excellent candidate for designing efficacious mucosal vaccines against other respiratory infections and for emergency preparedness against emerging epidemic and pandemic pathogens. IMPORTANCE According to the World Health Organization, COVID-19 may have caused ~15-million deaths across the globe and is still ravaging the world. Another wave of ~100 million infections is predicted in the United States due to the emergence of highly transmissible immune-escaped Omicron variants. The authorized vaccines would not prevent these transmissions since they do not trigger mucosal immunity. We circumvented this limitation by developing a needle-free, bacteriophage T4-based, mucosal vaccine. This intranasally administered vaccine generates superior mucosal immunity in mice, in addition to inducing robust humoral and cell-mediated immune responses, and provides complete protection and sterilizing immunity against SARS-CoV-2 variants. The vaccine is stable, adjuvant-free, and cost-effectively manufactured and distributed, making it a strategically important next-generation COVID vaccine for ending this pandemic.

Most gene-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are nonreplicating vectors. They deliver the gene or messenger RNA to the cell to express the spike protein but do not replicate to amplify antigen production. This study tested the utility of replication in a vaccine by comparing replication-defective adenovirus (RD-Ad) and replicating single-cycle adenovirus (SC-Ad) vaccines that express the SARS-CoV-2 spike protein. SC-Ad produced 100 times more spike protein than RD-Ad and generated significantly higher antibodies against the spike protein than RD-Ad after single immunization of Ad-permissive hamsters. SC-Ad-generated antibodies climbed over 14 weeks after single immunization and persisted for more than 10 months. When the hamsters were challenged 10.5 months after single immunization, a single intranasal or intramuscular immunization with SC-Ad-Spike reduced SARS-CoV-2 viral loads and damage in the lungs and preserved body weight better than vaccination with RD-Ad-Spike. This demonstrates the utility of harnessing replication in vaccines to amplify protection against infectious diseases.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of compounds, self-masked aldehyde inhibitors (SMAIs) which are based on the dipeptide aldehyde inhibitor (Cbz-Phe-Phe-CHO, 1 ), for which the P 1 Phe group contains a 1'-hydroxy group, effectively, an o -tyrosinyl aldehyde (Cbz-Phe- o -Tyr-CHO, 2 ; (Li et al. (2021) J. Med. Chem. 64, 11,267-11,287)). Compound 2 and other SMAIs exist in aqueous mixtures as stable δ-lactols, and apparent catalysis by the cysteine protease cruzain, the major cysteine protease of Trypanosoma cruzi , results in the opening of the lactol ring to afford the aldehydes which then form reversible thiohemiacetals with the enzyme. These SMAIs are also potent, time-dependent inhibitors of human cathepsin L ( K i = 11-60 nM), an enzyme which shares 36% amino acid identity with cruzain. As inactivators of cathepsin L have recently been shown to be potent anti-SARS-CoV-2 agents in infected mammalian cells (Mellott et al. (2021) ACS Chem. Biol. 16 , 642-650), we evaluated SMAIs in VeroE6 and A549/ACE2 cells infected with SARS-CoV-2. These SMAIs demonstrated potent anti-SARS-CoV-2 activity with values of EC 50 = 2-8 μM. We also synthesized pro-drug forms of the SMAIs in which the hydroxyl groups of the lactols were O-acylated. Such pro-drug SMAIs resulted in significantly enhanced anti-SARS-CoV-2 activity (EC 50 = 0.3-0.6 μM), demonstrating that the O-acylated-SMAIs afforded a level of stability within infected cells, and are likely converted to SMAIs by the action of cellular esterases. Lastly, we prepared and characterized an SMAI in which the sidechain adjacent to the terminal aldehyde is a 2-pyridonyl-alanine group, a mimic of both phenylalanine and glutamine. This compound ( 9 ) inhibited both cathepsin L and 3CL protease at low nanomolar concentrations, and also exerted anti-CoV-2 activity in an infected human cell line., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhu, Li, Drelich, Chenna, Mellott, Taylor, Tat, Garcia, Katzfuss, Tseng and Meek.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Dry heat decontamination has been shown to effectively inactivate viruses without compromising the integrity of delicate personal protective equipment (PPE), allowing safe reuse and helping to alleviate shortages of PPE that have arisen due to COVID-19. Unfortunately, current thermal decontamination guidelines rely on empirical data which are often sparse, limited to a specific virus, and unable to provide fundamental insight into the underlying inactivation reaction. In this work, we experimentally quantified dry heat decontamination of SARS-CoV-2 on disposable masks and validated a model that treats the inactivation reaction as thermal degradation of macromolecules. Furthermore, upon nondimensionalization, all of the experimental data collapse onto a unified curve, revealing that the thermally driven decontamination process exhibits self-similar behavior. Our results show that heating surgical masks to 70 °C for 5 min inactivates over 99.9% of SARS-CoV-2. We also characterized the chemical and physical properties of disposable masks after heat treatment and did not observe degradation. The model presented in this work enables extrapolation of results beyond specific temperatures to provide guidelines for safe PPE decontamination. The modeling framework and self-similar behavior are expected to extend to most viruses-including yet-unencountered novel viruses-while accounting for a range of environmental conditions., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus responsible for the ongoing COVID-19 pandemic. SARS-CoV-2 binds to the human cell receptor angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain in the S1 subunit of the spike protein (S1-RBD). The serum levels of autoantibodies against ACE2 are significantly higher in patients with COVID-19 than in controls and are associated with disease severity. However, the mechanisms through which these anti-ACE2 antibodies are induced during SARS-CoV-2 infection are unclear. In this study, we confirmed the increase in antibodies against ACE2 in patients with COVID-19 and found a positive correlation between the amounts of antibodies against ACE2 and S1-RBD. Moreover, antibody binding to ACE2 was significantly decreased in the sera of some COVID-19 patients after preadsorption of the sera with S1-RBD, which indicated that antibodies against S1-RBD can cross-react with ACE2. To confirm this possibility, two monoclonal antibodies (mAbs 127 and 150) which could bind to both S1-RBD and ACE2 were isolated from S1-RBD-immunized mice. Measurement of the binding affinities by Biacore showed these two mAbs bind to ACE2 much weaker than binding to S1-RBD. Epitope mapping using synthetic overlapping peptides and hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that the amino acid residues P463, F464, E465, R466, D467 and E471 of S1-RBD are critical for the recognition by mAbs 127 and 150. In addition, Western blotting analysis showed that these mAbs could recognize ACE2 only in native but not denatured form, indicating the ACE2 epitopes recognized by these mAbs were conformation-dependent. The protein-protein interaction between ACE2 and the higher affinity mAb 127 was analyzed by HDX-MS and visualized by negative-stain transmission electron microscopy imaging combined with antigen-antibody docking. Together, our results suggest that ACE2-cross-reactive anti-S1-RBD antibodies can be induced during SARS-CoV-2 infection due to potential antigenic cross-reactivity between S1-RBD and its receptor ACE2., Competing Interests: Y-CL was employed by Leadgene Biomedical, Inc. Y-WC, Y-YC, L-YC, Y-RW, and Y-CC are employed by Leadgene Biomedical, Inc. C-DC is employed by OmicsLab Co., Ltd. C-KT and C-KL are employed by SIDSCO Biomedical Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lai, Cheng, Chao, Chang, Chen, Tsai, Wang, Lin, Chang, Chuang, Chen, Wang, Chang, Huang, Wang, Tseng, Lin, Chuang and Yeh.)

Dengue virus (DENV) which infects about 390 million people per year in tropical and subtropical areas manifests various disease symptoms, ranging from fever to life-threatening hemorrhage and even shock. To date, there is still no effective treatment for DENV disease, but only supportive care. DENV nonstructural protein 1 (NS1) has been shown to play a key role in disease pathogenesis. Recent studies have shown that anti-DENV NS1 antibody can provide disease protection by blocking the DENV-induced disruption of endothelial integrity. We previously demonstrated that anti-NS1 monoclonal antibody (mAb) protected mice from all four serotypes of DENV challenge. Here, we generated humanized anti-NS1 mAbs and transferred them to mice after DENV infection. The results showed that DENV-induced prolonged bleeding time and skin hemorrhage were reduced, even several days after DENV challenge. Mechanistic studies showed the ability of humanized anti-NS1 mAbs to inhibit NS1-induced vascular hyperpermeability and to elicit Fcγ-dependent complement-mediated cytolysis as well as antibody-dependent cellular cytotoxicity of cells infected with four serotypes of DENV. These results highlight humanized anti-NS1 mAb as a potential therapeutic agent in DENV infection., Competing Interests: The authors have declared that no competing interests exist.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with angiotensin converting enzyme 2 (ACE2) interface, which enables 2G1 to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar half maximal inhibitory concentration in vitro. In SARS-CoV-2, Beta or Delta variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 is potentially capable of dealing with emerging SARS-CoV-2 variants in the future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy., (© 2022. The Author(s).)

Background: The current clinical workflow for esophageal gross tumor volume (GTV) contouring relies on manual delineation with high labor costs and inter-user variability., Purpose: To validate the clinical applicability of a deep learning multimodality esophageal GTV contouring model, developed at one institution whereas tested at multiple institutions., Materials and Methods: We collected 606 patients with esophageal cancer retrospectively from four institutions. Among them, 252 patients from institution 1 contained both a treatment planning CT (pCT) and a pair of diagnostic FDG-PET/CT; 354 patients from three other institutions had only pCT scans under different staging protocols or lacking PET scanners. A two-streamed deep learning model for GTV segmentation was developed using pCT and PET/CT scans of a subset (148 patients) from institution 1. This built model had the flexibility of segmenting GTVs via only pCT or pCT+PET/CT combined when available. For independent evaluation, the remaining 104 patients from institution 1 behaved as an unseen internal testing, and 354 patients from the other three institutions were used for external testing. Degrees of manual revision were further evaluated by human experts to assess the contour-editing effort. Furthermore, the deep model's performance was compared against four radiation oncologists in a multi-user study using 20 randomly chosen external patients. Contouring accuracy and time were recorded for the pre- and post-deep learning-assisted delineation process., Competing Interests: DG, LL, and DJ were employed by PAII Inc. YX, LH, GX, and JX are employed by Ping An Technology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ye, Guo, Tseng, Ge, Hung, Pai, Ren, Zheng, Zhu, Peng, Chen, Chen, Chou, Chen, Yu, Chen, Jiao, Xin, Huang, Xie, Xiao, Lu, Yan, Jin and Ho.)

Objective: To design and verify a CMOS 256-pixel photovoltaic-powered subretinal prosthetic chip with key advances over the state-of-the-art. The three key advances are: 1) automatic adaptation to changing background illuminance levels; 2) increase of injection charges with reduced crosstalk leakage charges, enhanced charge balance, and low process variations; 3) stable stimulation voltage to keep the safety of water window., Methods: The novel auto-adaptive pixel circuit is designed to realize the Michealis - Menten equation (MME) so that the automatic adaptation to changing background illuminance can be achieved. Both improved biphasic constant current stimulator (CCS) via bi-directional shared electrodes (BDSEs) with optimized stimulation pattern and improved constant current generator/ring oscillator are designed to achieve the above second advance on injection charges. The closed-loop charge pump is designed to achieve the third advance., Results: The measured dynamic range of image illuminance is increased to 54.7 dB. The maximum stimulation charge is 8.89nC. The measured stimulation current mismatch is 1.7% and the measured residual charge is 0.150 nC. The measured variations of stimulation frequencies are from 26 Hz to 29.7 Hz. The results of ex vivo tests have shown that the proposed subretinal chip can evoke spiking responses of RGCs. The function of adaptation process to background illuminance has also been verified., Conclusion and Significance: Through both electrical measurement and ex vivo tests, the functions of designed subretinal chip have been validated successfully. It is shown that the proposed subretinal chip is a promising solution for subretinal prostheses.

Dengue virus (DENV) is a cause of vascular endothelial dysfunction and vascular leakage, which are characterized as hallmarks of dengue hemorrhagic fever or dengue shock syndrome, which become a severe global health emergency with substantial morbidity and mortality. Currently, there are still no promising therapeutics to alleviate the dengue-associated vascular hemorrhage in a clinical setting. In the present study, we first observed that heme oxygenase-1 (HO-1) expression level was highly suppressed in severe DENV-infected patients. In contrast, the overexpression of HO-1 could attenuate DENV-induced pathogenesis, including plasma leakage and thrombocytopenia, in an AG129 mouse model. Our data indicate that overexpression of HO-1 or its metabolite biliverdin can maintain endothelial integrity upon DENV infection in vitro and in vivo. We further characterized the positive regulatory effect of HO-1 on the endothelial adhesion factor vascular endothelial-cadherin to decrease DENV-induced endothelial hyperpermeability. Subsequently, we confirmed that two medicinal plant-derived compounds, andrographolide, and celastrol, widely used as a nutritional or medicinal supplement are useful to attenuate DENV-induced plasma leakage through induction of the HO-1 expression in DENV-infected AG129 mice. In conclusion, our findings reveal that induction of the HO-1 signal pathway is a promising option for the treatment of DENV-induced vascular pathologies., (© 2021 Federation of American Societies for Experimental Biology.)

We report the discovery of several highly potent small molecules with low-nM potency against severe acute respiratory syndrome coronavirus (SARS-CoV; lowest half-maximal inhibitory concentration (IC 50 : 13 nM), SARS-CoV-2 (IC 50 : 23 nM), and Middle East respiratory syndrome coronavirus (MERS-CoV; IC 50 : 76 nM) in pseudovirus-based assays with excellent selectivity index (SI) values (>5000), demonstrating potential pan-coronavirus inhibitory activities. Some compounds showed 100% inhibition against the cytopathic effects (CPE; IC 100 ) of an authentic SARS-CoV-2 (US&#95;WA-1/2020) variant at 1.25 µM. The most active inhibitors also potently inhibited variants of concern (VOCs), including the UK (B.1.1.7) and South African (B.1.351) variants and the Delta variant (B.1.617.2) originally identified in India in pseudovirus-based assay. Surface plasmon resonance (SPR) analysis with one potent inhibitor confirmed that it binds to the prefusion SARS-CoV-2 spike protein trimer. These small-molecule inhibitors prevented virus-mediated cell-cell fusion. The absorption, distribution, metabolism, and excretion (ADME) data for one of the most active inhibitors, NBCoV1, demonstrated drug-like properties. An in vivo pharmacokinetics (PK) study of NBCoV1 in rats demonstrated an excellent half-life (t 1/2 ) of 11.3 h, a mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect these lead inhibitors to facilitate the further development of preclinical and clinical candidates.

Asunercept (company code APG101 [Apogenix AG]; company code CAN008 [CANbridge Pharmaceuticals]) is a novel glycosylated fusion protein that has shown promising effectiveness in glioblastoma. This Phase I study was initiated to evaluate the tolerability and safety of asunercept in combination with standard radiotherapy and temozolomide (RT/TMZ) in Asian patients with newly diagnosed glioblastoma. This was the Phase I portion of a Phase I/II open label, multicenter trial of asunercept plus standard RT/TMZ. Adults with newly-diagnosed glioblastoma received surgical resection followed by standard RT/TMZ plus asunercept 200 mg/week (Cohort 1) or 400 mg/week (Cohort 2) by 30-min IV infusion. The primary endpoint was the safety and tolerability of asunercept during concurrent asunercept and RT/TMZ; dose-limiting toxicities were observed for each dose. Secondary endpoints included pharmacokinetics (PK) and 6-month progression-free survival (PFS6). All patients (Cohort 1, n = 3; Cohort 2, n = 7) completed ≥ 7 weeks of asunercept treatment. No DLTs were experienced. Only one possibly treatment-related treatment emergent adverse event (TEAE), Grade 1 gingival swelling, was observed. No Grade > 3 TEAEs were reported and no TEAE led to treatment discontinuation. Systemic asunercept exposure increased proportionally with dose and showed low inter-patient variability. The PFS6 rate was 33.3% and 57.1% for patients in Cohort 1 and 2, respectively. Patients in Cohort 2 maintained a PFS rate of 57.1% at Month 12. Adding asunercept to standard RT/TMZ was safe and well tolerated in patients with newly-diagnosed glioblastoma and 400 mg/week resulted in encouraging efficacy.Trial registration NCT02853565, August 3, 2016., (© 2021. The Author(s).)

Background/aim: Cisplatin with 5-fluouracil (Cis/5Fu) and paclitaxel with carboplatin (Pac/Car) are common regimens used in concurrent chemoradiotherapy (CCRT) for patients with locally advanced esophageal cancer (EC). Here, we aimed to compare the survival outcomes and treatment-related toxicities between these regimens in neoadjuvant CCRT in patients with locally advanced EC., Patients and Methods: One hundred and thirty-six patients with locally advanced EC (98% squamous cell carcinoma) were prospectively recruited between 2016 and 2017 in a non-randomized manner. Patients were categorized into two groups according to the chemotherapeutic agents administered (Pac/Car group, n=87; Cis/5Fu group, n=47) in CCRT to compare the survival outcome and severe adverse event (sAE) incidence., Results: Forty-two patients (85.7%) and 80 patients (91.4%) in the Cis/5Fu and Pac/Car groups completed pre-planned CCRT (p=0.26), respectively. The Cis/5Fu group presented a higher incidence of non-hematological sAE than the Pac/Car group (69.45% vs. 51.7%, p=0.049). Patients in the Pac/Car group showed a higher rate of surgical resection than those in the Cis/5Fu group (49.4% vs. 22.4%, p<0.001). After a median follow-up duration of 22.0 months (range=1.9-31.8), the 2-year survival rate was 56.9% for patients in the Pac/Car group and 28.7% for the Cis/5Fu group. The hazard ratio (HR) of overall survival was 0.45 (95%CI=0.28-0.72, p=0.001) in the comparison between the groups., Conclusion: Overall, neoadjuvant CCRT with Pac/Car is associated with a better survival outcome, higher surgical resection rate, and better safety profiles than Cis/5Fu in patients with locally advanced EC., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The clinical significance of frailty status on treatment outcome in patients with esophageal cancer (EC) has been seldom explored. This study aimed to evaluate the impact of pretreatment frailty on treatment-related toxicity and survival outcome in patients with EC undergoing concurrent chemoradiotherapy (CCRT)., Patients and Methods: Patients aged ≥20 years and with newly diagnosed locally advanced EC receiving neoadjuvant radiotherapy and concurrent chemotherapy with weekly administration of carboplatin and paclitaxel for 5 weeks were prospectively enrolled. A pretreatment frailty assessment was performed within 7 days before CCRT initiation. The primary endpoint was treatment-related toxicity and complications of CCRT while the secondary endpoint was overall survival., Results: A total of 87 patients were enrolled, 41 (47%) and 46 (53%) of whom were allocated in the frail and fit group, respectively. Frail patients had a significantly higher incidence of having at least one severe hematological adverse event (63.4% vs. 19.6%, p<0.001), higher risk of emergent room visiting [relative risk 3.72; 95% confidence interval (CI)=1.39-9.91; p=0.009] and hospitalization (relative risk 3.85; 95% CI=1.03-11.2; p=0.013) during the course of CCRT, when compared to fit patients. Overall survival showed significant worsening in the frail group [adjusted hazard ratio (HR)=2.12; 95% CI=1.01-4.42; p=0.046]., Conclusion: Frailty is associated with increase of treatment-related toxicities and poor survival outcome in EC patients undergoing CCRT. Our study suggested that pretreatment frailty assessment is imperative to serve as a predictor and prognostic factor for all adult patients with EC undergoing CCRT., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: To investigate the clinical utility of short-course induction chemotherapy followed by low-dose radiotherapy without a tumor bed boost in patients with primary central nervous system (CNS) germinomas., Methods: We retrospectively reviewed the clinical records of patients with primary CNS germinomas who received short-course induction chemotherapy (2 cycles of cisplatin 20 mg/m2 plus etoposide 40 or 100 mg/m2 for 5 days) followed by low-dose radiotherapy (dose: 2340 cGy) without a tumor bed boost. Disease-free survival and overall survival served as the main outcome measures., Results: Between February 2002 and June 2018, 24 patients (20 males and 4 females; median age: 14.1 y; age range: 7.9 to 21.2 y) with pathology-proven CNS germinomas were included. The median follow-up time was 106 months (range: 17 to 169 mo). Isolated and multifocal lesions were identified in 13 and 11 patients, respectively. Tumor location was as follows: pineal gland (n=17), suprasellar region (n=13), periventricular region (n=7), and basal ganglia (n=2). Five patients had increased levels (>5 mIU/mL) of beta-human chorionic gonadotropin (β-hCG), whereas alpha-fetoprotein concentrations were within the reference range in all participants. A total of 16 patients achieved remission after induction chemotherapy. The complete response rates of patients with increased and normal β-hCG levels were 40.0% and 72.2%, respectively (P=0.208). Low-dose radiotherapy without a tumor bed boost was subsequently delivered to either the whole ventricle (n=16) or the whole brain (n=8), resulting in complete remission in all participants. Compared with patients without increased β-hCG levels, those with β-hCG-secreting germinomas had less favorable 5-year disease-free survival rates (100% vs. 60%, respectively, P=0.000115)., Conclusions: Some children with primary CNS germinoma may benefit from short-course induction chemotherapy followed by low-dose radiotherapy to the whole ventricle without a tumor bed boost. The validity of our findings needs to be confirmed in a randomized phase II study for children with β-hCG levels <5 mIU/mL., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The c.G6055A (p.G2019S) mutation in leucine-rich repeat kinase 2 (LRRK2) is the most prevalent genetic cause of Parkinson's disease (PD). CRISPR/Cas9-mediated genome editing by homology-directed repair (HDR) has been applied to correct the mutation but may create small insertions and deletions (indels) due to double-strand DNA breaks. Adenine base editors (ABEs) could convert targeted A·T to G·C in genomic DNA without double-strand breaks. However, the correction efficiency of ABE in LRRK2 c.G6055A (p.G2019S) mutation remains unknown yet. This study aimed to compare the mutation correction efficiencies and off-target effects between HDR and ABEs in induced pluripotent stem cells (iPSCs) carrying LRRK2 c.G6055A (p.G2019S) mutation., Methods: A set of mutation-corrected isogenic lines by editing the LRRK2 c.G6055A (p.G2019S) mutation in a PD patient-derived iPSC line using HDR or ABE were established. The mutation correction efficacies, off-target effects, and indels between HDR and ABE were compared. Comparative transcriptomic and proteomic analyses between the LRRK2 p.G2019S iPSCs and isogenic control cells were performed to identify novel molecular targets involved in LRRK2-parkinsonism pathways., Results: ABE had a higher correction rate (13/53 clones, 24.5%) than HDR (3/47 clones, 6.4%). Twenty-seven HDR clones (57.4%), but no ABE clones, had deletions, though 14 ABE clones (26.4%) had off-target mutations. The corrected isogenic iPSC-derived dopaminergic neurons exhibited reduced LRRK2 kinase activity, decreased phospho-α-synuclein expression, and mitigated neurite shrinkage and apoptosis. Comparative transcriptomic and proteomic analysis identified different gene expression patterns in energy metabolism, protein degradation, and peroxisome proliferator-activated receptor pathways between the mutant and isogenic control cells., Conclusions: The results of this study envision that ABE could directly correct the pathogenic mutation in iPSCs for reversing disease-related phenotypes in neuropathology and exploring novel pathophysiological targets in PD., (© 2021. The Author(s).)

A “universal” platform that can rapidly generate multiplex vaccine candidates is critically needed to control pandemics. Using the severe acute respiratory syndrome coronavirus 2 as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates was engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers was found to be the most potent vaccine candidate in animal models. Without any adjuvant, this vaccine stimulated robust immune responses, both T helper cell 1 (T H 1) and T H 2 immunoglobulin G subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new nanovaccine design framework might allow the rapid deployment of effective adjuvant-free phage-based vaccines against any emerging pathogen in the future.

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: The University of Minnesota has filed a patent on the lumazine synthase nanoparticle-based SARS-CoV-2 RBD vaccine with F.L, Q.G., Y.W., J.S., M.J., S.H., L.D. and T.W. as inventors. Other authors have declared that no competing interests exist.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease- Trypanosoma cruzi . These SMAIs exerted potent, reversible inhibition of cruzain ( K i * = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

Although SARS-CoV-2-neutralizing antibodies are promising therapeutics against COVID-19, little is known about their mechanism(s) of action or effective dosing windows. We report the generation and development of SC31, a potent SARS-CoV-2 neutralizing antibody, isolated from a convalescent patient. Antibody-mediated neutralization occurs via an epitope within the receptor-binding domain of the SARS-CoV-2 Spike protein. SC31 exhibited potent anti-SARS-CoV-2 activities in multiple animal models. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, treatment with SC31 greatly reduced viral loads and attenuated pro-inflammatory responses linked to the severity of COVID-19. Importantly, a comparison of the efficacies of SC31 and its Fc-null LALA variant revealed that the optimal therapeutic efficacy of SC31 requires Fc-mediated effector functions that promote IFNγ-driven anti-viral immune responses, in addition to its neutralization ability. A dose-dependent efficacy of SC31 was observed down to 5mg/kg when administered before viral-induced lung inflammatory responses. In addition, antibody-dependent enhancement was not observed even when infected mice were treated with SC31 at sub-therapeutic doses. In SARS-CoV-2-infected hamsters, SC31 treatment significantly prevented weight loss, reduced viral loads, and attenuated the histopathology of the lungs. In rhesus macaques, the therapeutic potential of SC31 was evidenced through the reduction of viral loads in both upper and lower respiratory tracts to undetectable levels. Together, the results of our preclinical studies demonstrated the therapeutic efficacy of SC31 in three different models and its potential as a COVID-19 therapeutic candidate., Competing Interests: B.J.H., C.E.Z.C., A.P.C.L., P.J.I., J.D.B-K., S.G. and D.T. are currently listed as inventors on patent application 10202010008W SARS-CoV-2 Spike Protein Antigen-Binding Molecules. J.D.B-K., S.G., D.T., and P.J.I. are affiliated with Hummingbird Bioscience (https://hummingbirdbioscience.com/)

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M Pro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M Pro ), we have designed and synthesized a series of SC2M Pro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M Pro active-site cysteine C145. All inhibitors display high potency with K i values at or below 100 nM. The most potent compound, MPI3, has as a K i value of 8.3 nM. Crystallographic analyses of SC2M Pro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 μM and A549/ACE2 cells at 0.16-0.31 μM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2M Pro inhibitors with ultra-high antiviral potency., (© 2020 Wiley-VCH GmbH.)

Guided by a computational docking analysis, about 30 Food and Drug Administration/European Medicines Agency (FDA/EMA)-approved small-molecule medicines were characterized on their inhibition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M p ro ). Of these small molecules tested, six displayed a concentration that inhibits response by 50% (IC 50 ) value below 100 μM in inhibiting M p ro , and, importantly, three, that is, pimozide, ebastine, and bepridil, are basic molecules that potentiate dual functions by both raising endosomal pH to interfere with SARS-CoV-2 entry into the human cell host and inhibiting M p ro in infected cells. A live virus-based modified microneutralization assay revealed that bepridil possesses significant anti-SARS-CoV-2 activity in both Vero E6 and A459/ACE2 cells in a dose-dependent manner with low micromolar effective concentration, 50% (EC 50 ) values. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Gross tumor volume (GTV) and clinical target volume (CTV) delineation are two critical steps in the cancer radiotherapy planning. GTV defines the primary treatment area of the gross tumor, while CTV outlines the sub-clinical malignant disease. Automatic GTV and CTV segmentation are both challenging for distinct reasons: GTV segmentation relies on the radiotherapy computed tomography (RTCT) image appearance, which suffers from poor contrast with the surrounding tissues, while CTV delineation relies on a mixture of predefined and judgement-based margins. High intra- and inter-user variability makes this a particularly difficult task. We develop tailored methods solving each task in the esophageal cancer radiotherapy, together leading to a comprehensive solution for the target contouring task. Specifically, we integrate the RTCT and positron emission tomography (PET) modalities together into a two-stream chained deep fusion framework taking advantage of both modalities to facilitate more accurate GTV segmentation. For CTV segmentation, since it is highly context-dependent-it must encompass the GTV and involved lymph nodes while also avoiding excessive exposure to the organs at risk-we formulate it as a deep contextual appearance-based problem using encoded spatial distances of these anatomical structures. This better emulates the margin- and appearance-based CTV delineation performed by oncologists. Adding to our contributions, for the GTV segmentation we propose a simple yet effective progressive semantically-nested network (PSNN) backbone that outperforms more complicated models. Our work is the first to provide a comprehensive solution for the esophageal GTV and CTV segmentation in radiotherapy planning. Extensive 4-fold cross-validation on 148 esophageal cancer patients, the largest analysis to date, was carried out for both tasks. The results demonstrate that our GTV and CTV segmentation approaches significantly improve the performance over previous state-of-the-art works, e.g., by 8.7% increases in Dice score (DSC) and 32.9mm reduction in Hausdorff distance (HD) for GTV segmentation, and by 3.4% increases in DSC and 29.4mm reduction in HD for CTV segmentation., Competing Interests: Declaration of Competing Interest We have no conflicts of interest to disclose., (Copyright © 2020. Published by Elsevier B.V.)

Heatstroke (HS) is an acute clinical disease characterized by abnormal hyperthermia and multi-organ dysfunction. Heme oxygenase (HO)-1, also called heat shock protein (HSP)32, is induced by hyperthermia and also plays protective roles in many lung disease models. Based on this phenomenon, we investigated the protective role of endogenous HO-1 in heat-induced lung damage in rats. Male Sprague-Dawley (SD) rats were separated into three groups: (a) normothermic sham, (b) HS, and (c) SnPP (inhibitor of HO-1) pretreatment rats. In the HS group, rats were killed at various time points (1, 3, 6, and 12 h after heat exposure) in order to analyze messenger ribonucleic acid (mRNA) and protein levels. Lung sections were examined for tissue damage and localization of HO-1 using immunofluorescence double labeling. We found that HS induced lung pathology (congested and thickened lung septa). The level of HO-1 mRNA was increased at 1 h, and the protein level peaked at 6 h after heat exposure. Pretreatment with SnPP (tin-protoporphyrin IX, 30 mg/kg, intraperitoneal injection for 1 h before heat exposure) aggravated the lung damage. Furthermore, we demonstrated HO-1 expression in lung type II pneumocytes. Our results suggest that endogenous HO-1 is protective against HS-induced lung damage. Induction of HO-1 may be a potential therapeutic strategy for treating heat-related diseases.