Resistance training combined with ß-hydroxy ß-methylbutyrate for patients with critical illness: A four-arm, mixed-methods, feasibility randomised controlled trial.

OBJECTIVES: This study aimed to assess the feasibility, safety, acceptability, and potential effectiveness of resistance training (RT) with or without ß-Hydroxy ß-Methylbutyrate (HMB) intervention program for ICU patients. DESIGN: Open-label, parallel group, mixed method, randomized controlled trial. SETTINGS: A tertiary general hospital in Fuzhou, China. METHODS: Participants were randomly allocated to one of four groups. The RT group received supervised multilevel resistance training (RT) using elastic bands, administered by trained ICU nurses. The HMB group received an additional daily dose of 3.0 g HMB. The combination group underwent both interventions concurrently, while the control group received standard care. These interventions were implemented throughout the entire hospitalization period. Primary outcomes included feasibility indicators such as recruitment rate, enrollment rate, retention rate, and compliance rate. Secondary outcomes covered adverse events, acceptability (evaluated through questionnaires and qualitative interviews), and physical function. Quantitative analysis utilized a generalized estimation equation model, while qualitative analysis employed directed content analysis. RESULTS: All feasibility indicators met predetermined criteria. Forty-eight patients were randomly assigned across four arms, achieving a 96% enrollment rate. Most patients adhered to the intervention until discharge, resulting in a 97.9% retention rate. Compliance rates for both RT and HMB interventions approached or exceeded 85%. No adverse events were reported. The intervention achieved 100% acceptability, with a prevailing expression of positive experiences and perception of appropriateness. The RT intervention shows potential improvement in physical function, while HMB does not. CONCLUSIONS: Implementing nurse-led resistance training with elastic bands with or without HMB proved to be feasible and safe for ICU patients. IMPLICATIONS FOR CLINICAL PRACTICE: A large-scale, multicenter clinical trials are imperative to definitively assess the impact of this intervention on functional outcomes in this population.

Effects of a multilevel intervention of resistance training with or without beta-hydroxy-beta-methylbutyrate in medical ICU patients during entire hospitalisation: a four-arm multicentre randomised controlled trial.

BACKGROUND: Intensive care unit-acquired weakness (ICU-AW) is a prevalent and severe issue among ICU patients. Resistance training and beta-hydroxy-beta-methylbutyrate (HMB) intervention have demonstrated the potential to enhance muscle function in patients with sarcopenia and in older adults. The purpose of this study was to determine whether resistance training and/or HMB administration would improve physical function, muscle strength, and quality of life in medical ICU patients. METHODS: In this multicentre, four-arm, single-blind randomised control trial, a total of 112 adult patients with internal medical diagnoses admitted to the ICU were enrolled. These participants were then randomly assigned to one of four treatment groups: the resistance training group received protocol-based multilevel resistance exercise, the HMB group received 3 g/day of HMBCa, combination group and control groups received standard care, from the ICU to the general ward until discharge. The primary outcomes assessed at discharge included six-minute walking distance (6MWD) and short physical performance battery (SPPB). Secondary outcomes measured included muscle mass, MRC score, grip strength, and health reports quality of life at different time points. Data analysis was performed using a generalised linear mixed model, adhering to the principles of intention-to-treat analysis. RESULTS: Resistance training and combination treatment groups exhibited significant increases in SPPB scores (3.848 and 2.832 points, respectively) compared to the control group and substantial improvements in 6WMD (99.768 and 88.577 m, respectively) (all with P < 0.01). However, no significant changes were observed in the HMB group. Muscle strength, as indicated by MRC and grip strength tests conducted at both ICU and hospital discharge, showed statistically significant improvements in the resistance training and combination groups (P < 0.05). Nevertheless, no significant differences were found between the treatment groups and usual care in terms of 60-day mortality, prevalence of ICU-AW, muscle mass, quality of life, or other functional aspects. CONCLUSIONS: Resistance training with or without beta-hydroxy-beta-methylbutyrate during the entire hospitalisation intervention improves physical function and muscle strength in medical ICU patients, but muscle mass, quality of life, and 60-day mortality were unaffected. TRIAL REGISTRATION: ChiCTR2200057685 was registered on March 15th, 2022.

Role of Microglia, Decreased Neurogenesis and Oligodendrocyte Depletion in Long COVID-Mediated Brain Impairments.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a recent worldwide coronavirus disease-2019 (COVID-19) pandemic. SARS-CoV-2 primarily causes an acute respiratory infection but can progress into significant neurological complications in some. Moreover, patients with severe acute COVID-19 could develop debilitating long-term sequela. Long-COVID is characterized by chronic symptoms that persist months after the initial infection. Common complaints are fatigue, myalgias, depression, anxiety, and "brain fog," or cognitive and memory impairments. A recent study demonstrated that a mild COVID-19 respiratory infection could generate elevated proinflammatory cytokines and chemokines in the cerebral spinal fluid. This commentary discusses findings from this study, demonstrating that even a mild respiratory SARS-CoV-2 infection can cause considerable neuroinflammation with microglial and macrophage reactivity. Such changes could also be gleaned by measuring chemokines and cytokines in the circulating blood. Moreover, neuroinflammation caused by mild SARS-CoV-2 infection can also impair hippocampal neurogenesis, deplete oligodendrocytes, and decrease myelinated axons. All these changes likely contribute to cognitive deficits in long-COVID syndrome. Therefore, strategies capable of restraining neuroinflammation, maintaining better hippocampal neurogenesis, and preserving oligodendrocyte lineage differentiation and maturation may prevent or reduce the incidence of long-COVID after SARS-CoV-2 respiratory infection.

Complications of COVID-19 on the Central Nervous System: Mechanisms and Potential Treatment for Easing Long COVID.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades human cells by binding to the angiotensin-converting-enzyme-2 (ACE-2) using a spike protein and leads to Coronavirus disease-2019 (COVID-19). COVID-19 primarily causes a respiratory infection that can lead to severe systemic inflammation. It is also common for some patients to develop significant neurological and psychiatric symptoms. The spread of SARS-CoV-2 to the CNS likely occurs through several pathways. Once spread in the CNS, many acute symptoms emerge, and such infections could also transpire into severe neurological complications, including encephalitis or ischemic stroke. After recovery from the acute infection, a significant percentage of patients develop "long COVID," a condition in which several symptoms of COVID-19 persist for prolonged periods. This review aims to discuss acute and chronic neurological problems after SARS-CoV-2 infection. The potential mechanisms by which SARS-CoV-2 enters the CNS and causes neuroinflammation, neuropathological changes observed in post-mortem brains of COVID-19 patients, and cognitive and mood problems in COVID-19 survivors are discussed in the initial part. The later part of the review deliberates the causes of long COVID, approaches for noninvasive tracking of neuroinflammation in long COVID patients, and the potential therapeutic strategies that could ease enduring CNS symptoms observed in long COVID.

Potential role of serum hypoxia-inducible factor 1alpha as a biomarker of delayed cerebral ischemia and poor clinical outcome after human aneurysmal subarachnoid hemorrhage: A prospective, longitudinal, multicenter, and observational study.

Objective: Hypoxia-inducible factor 1alpha (HIF-1α) functions as a crucial transcriptional mediator in hypoxic and ischemic brain response. We endeavored to assess the prognostic significance of serum HIF-1α in human aneurysmal subarachnoid hemorrhage (aSAH). Methods: In this prospective, longitudinal, multicenter, and observational study of 257 patients with aSAH and 100 healthy controls, serum HIF-1α levels were quantified. Univariate analyses, followed by multivariate analyses, were performed to discern the relationship between serum HIF-1α levels and severity and delayed cerebral ischemia (DCI) plus poststroke 6-month poor outcome [extended Glasgow outcome scale (GOSE) scores of 1-4]. Predictive efficiency was determined under the receiver operating characteristic (ROC) curve. Results: There were significantly increased serum HIF-lα levels after aSAH, in comparison to controls (median, 288.0 vs. 102.6 pg/ml; P < 0.001). Serum HIF-lα levels were independently correlated with Hunt-Hess scores [ß, 78.376; 95% confidence interval (CI): 56.446-100.305; P = 0.001] and modified Fisher scores (ß, 52.037; 95% CI: 23.461-80.614; P = 0.002). Serum HIF-lα levels displayed significant efficiency for discriminating DCI risk [area under ROC curve (AUC), 0.751; 95% CI: 0.687-0.815; P < 0.001] and poor outcome (AUC, 0.791; 95% CI: 0.736-0.846; P < 0.001). Using the Youden method, serum HIF-1α levels >229.3 pg/ml predicted the development of DCI with 92.3% sensitivity and 48.4% specificity and serum HIF-1α levels >384.0 pg/ml differentiated the risk of a poor prognosis with 71.4% sensitivity and 81.1% specificity. Serum HIF-1α levels >229.3 pg/ml were independently predictive of DCI [odds ratio (OR), 3.061; 95% CI: 1.045-8.965; P = 0.041] and serum HIF-1α levels >384.0 pg/ml were independently associated with a poor outcome (OR, 2.907; 95% CI: 1.403-6.024; P = 0.004). The DCI predictive ability of their combination was significantly superior to those of Hunt-Hess scores (AUC, 0.800; 95% CI: 0.745-0.855; P = 0.039) and modified Fisher scores (AUC, 0.784; 95% CI: 0.726-0.843; P = 0.004). The prognostic predictive ability of their combination substantially exceeded those of Hunt-Hess scores (AUC, 0.839; 95% CI: 0.791-0.886; P < 0.001) and modified Fisher scores (AUC, 0.844; 95% CI: 0.799-0.890; P < 0.001). Conclusion: Elevated serum HIF-lα levels after aSAH, in independent correlation with stroke severity, were independently associated with DCI and 6-month poor outcome, substantializing serum HIF-lα as a potential prognostic biomarker of aSAH.

Cinobufacini Inhibits the Development of Pancreatic Cancer Cells through the TGFß/Smads Pathway of Pancreatic Stellate Cells.

This study aimed to test cinobufacini therapeutic potential for pancreatic cancer, verify its potential molecular mechanism, and evaluate the cinobufacini impact on pancreatic cancer microenvironment. First, the effect of cinobufacini-treated pancreatic stellate cells (PSCs) supernatant on the value-added ability of pancreatic cancer (PCCs) was tested. The results show that cinobufacini can effectively reduce the ability of PSCs supernatant to promote the value-added PCCs. Further results show that cinobufacini can effectively reduce the concentration of TGFß in the supernatant of PSCs. Subsequently, the impact of cinobufacini on the transcription and translation levels of key genes in the TGFß/Smads pathway was examined. The results showed that the impact of cinobufacini on the transcription levels of Smad2, Smad3, and Smad7 was in a concentration-dependent manner, while the transcriptional activity of collagen I mRNA was decreased with the increase of cinobufacini concentration. The results of protein expression showed that cinobufacini could upregulate the expression of inhibitory protein Smad7, inhibit the phosphorylation level of p-Smad2/3, and then suppress the expression of type I collagen (collagen I). On the one hand, this study shows that cinobufacini can inhibit the promotion of PSCs on the proliferation of PCCs. On the other hand, cinobufacini can upregulate the expression of the inhibitory molecule, Smad7, through the TGFß/Smads pathway and reduce the phosphorylation level of p-Smad2/3, thereby inhibiting the expression of collagen I and pancreatic fibrosis. cinobufacin can inhibit the proliferation of SW1900 cells by blocking the TGFß/Smads pathway of pancreatic stellate cells. These results provide a clinical basis for the treatment of pancreatic cancer.

Can mild cognitive impairment and Alzheimer's disease be diagnosed by monitoring a miRNA triad in the blood?

Objectively diagnosing age-related cognitive impairment (ACI), mild cognitive impairment (MCI), and early-stage Alzheimer's disease (AD) is a difficult task, as most cognitive impairment is clinically established via questionnaires, history, and physical examinations. A recent study has suggested that monitoring a miRNA triad, miR-181a-5p, miR-146a-5p, and miR-148a-3p can identify ACI and its progression to MCI and AD (Islam et al., EMBO Mol Med. 13: e14997, 2021). This commentary deliberates findings from this article, such as elevated levels of the miRNA triad in the brain impairing neural plasticity and cognitive function, the efficiency of measuring the miRNA triad in the circulating blood diagnosing MCI and AD, and the promise for improving cognitive function in MCI and AD by inhibiting this miRNA triad. Additional studies required prior to employing this miRNA triad in clinical practice are also discussed.

Positive feedback regulation of lncRNA PVT1 and HIF2α contributes to clear cell renal cell carcinoma tumorigenesis and metastasis.

Long noncoding RNAs (lncRNAs) have been reported to exert important roles in tumors, including clear cell renal cell carcinoma (ccRCC). PVT1 is an important oncogenic lncRNA which has critical effects on onset and development of various cancers, however, the underlying mechanism of PVT1 functioning in ccRCC remains largely unknown. VHL deficiency-induced HIF2α accumulation is one of the major factors for ccRCC. Here, we identified the potential molecular mechanism of PVT1 in promoting ccRCC development by stabilizing HIF2α. PVT1 was significantly upregulated in ccRCC tissues and high PVT1 expression was associated with poor prognosis of ccRCC patients. Both gain-of-function and loss-of function experiments revealed that PVT1 enhanced ccRCC cells proliferation, migration, and invasion and induced tumor angiogenesis in vitro and in vivo. Mechanistically, PVT1 interacted with HIF2α protein and enhanced its stability by protecting it from ubiquitination-dependent degradation, thereby exerting its biological significance. Meanwhile, HIF2α bound to the enhancer of PVT1 to transactivate its expression. Furthermore, HIF2α specific inhibitor could repress PVT1 expression and its oncogenic functions. Therefore, our study demonstrates that the PVT1/ HIF2α positive feedback loop involves in tumorigenesis and progression of ccRCC, which may be exploited for anticancer therapy.

Serum Hypoxia-Inducible Factor 1alpha Levels Correlate with Outcomes After Intracerebral Hemorrhage.

BACKGROUND: Serum hypoxia-inducible factor 1alpha (HIF-1α) is a key regulator in hypoxic and ischemic brain injury. We determined the relationship between serum HIF-1α levels and long-term prognosis plus severity of intracerebral hemorrhage (ICH). METHODS: A total of 97 ICH cases and 97 healthy controls were enrolled. Glasgow Coma Scale (GCS) score and hematoma volume were used to assess hemorrhagic severity. Glasgow Outcome Scale (GOS) score of 1-3 at post-stroke 90 days was defined as a poor outcome. RESULTS: Serum HIF-1α levels of ICH patients were significantly higher than those of healthy controls (median, 218.8 vs 105.4 pg/mL; P<0.001) and were substantially correlated with GCS score (r=-0.485, P<0.001), hematoma volume (r=0.357, P<0.001) and GOS score (r=-0.436, P<0.001). Serum HIF-1α levels >239.4 pg/mL discriminated patients at risk of 90-day poor outcome with sensitivity of 65.9% and specificity of 79.3% (area under the receiver operating characteristic curve, 0.725; 95% confidence interval, 0.625-0.811; P<0.001). Moreover, serum HIF-1α levels >239.4 pg/mL were independently associated with a poor 90-day outcome (odds ratio, 5.133; 95% confidence interval, 1.117-23.593; P=0.036). CONCLUSION: Serum HIF-1α, in close correlation with hemorrhagic severity and poor 90-day outcome, may serve as a potential prognostic biomarker for ICH.

Treating Parkinson's disease by astrocyte reprogramming: Progress and challenges.

Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, is typified by both motor and nonmotor symptoms. The current medications provide symptomatic relief but do not stimulate the production of new dopaminergic neurons in the substantia nigra. Astrocyte reprogramming has recently received much attention as an avenue for increasing functional dopaminergic neurons in the mouse PD brain. By targeting a microRNA (miRNA) loop, astrocytes in the mouse brain could be reprogrammed into functional dopaminergic neurons. Such in vivo astrocyte reprogramming in the mouse model of PD has successfully added new dopaminergic neurons to the substantia nigra and increased dopamine levels associated with axonal projections into the striatum. This review deliberates the astrocyte reprogramming methods using specific transcription factors and mRNAs and the progress in generating dopaminergic neurons in vivo. In addition, the translational potential, challenges, and potential risks of astrocyte reprogramming for an enduring alleviation of parkinsonian symptoms are conferred.

Serum glucose and potassium ratio as a predictive factor for prognosis of acute intracerebral hemorrhage.

OBJECTIVE: The serum glucose/potassium ratio (GPR) is a potential prognostic predictor for acute brain injury-related diseases. We calculated the serum GPR in patients with acute intracerebral hemorrhage (ICH) and explored its prognostic value for long-term prognoses and ICH severity. METHODS: This retrospective cohort study consecutively included 92 patients with ICH and 92 healthy controls. The National Institutes of Health Stroke Scale (NIHSS) score, Glasgow coma scale (GCS) score, and hematoma volume were used to assess severity. A modified Rankin Scale score > 2 at 90 days post-stroke was defined as a poor outcome. RESULTS: The serum GPR was significantly higher in patients than controls. The serum GPR was weakly correlated with the NIHSS score, GCS score, and hematoma volume. The serum GPR, GCS score, and hematoma volume were independently associated with poor outcomes. In the receiver operating characteristic curve analysis, the serum GPR remarkably discriminated patients at risk of poor outcomes at 90 days. The serum GPR significantly improved the prognostic predictive capability of hematoma volume and tended to increase that of the GCS score. CONCLUSION: Serum GPR is an easily obtained clinical variable for predicting clinical outcomes after ICH.

Assessment of acute pancreatitis severity via determination of serum levels of hsa-miR-126-5p and IL-6.

Early assessment of acute pancreatitis (AP) severity is key to its treatment. The present study aimed to explore the role of microRNAs (miRNAs/miRs) combined with inflammatory factors in determining AP severity. For this, serum pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-8 and IL-10)] and miRNAs [Homo sapiens (hsa)-miR-548d-5p, hsa-miR-126-5p and hsa-miR-130b-5p] were detected in patients with mild AP (MAP), severe AP (SAP) and recurrent AP (RAP). High expression of IL-10, TNF-α, hsa-miR-126-5p, hsa-miR-548d-5p and hsa-miR-130b-5p was able to distinguish SAP from MAP and RAP (P<0.05). Multifactorial binary logistic regression analysis indicated that IL-1/IL-6 combined with hsa-miR-126-5p/hsa-miR-548d-5p had a significant influence on AP and AP severity (P<0.05). Receiver operating characteristic analysis revealed that IL-1 combined with hsa-miR-126-5p [area under the curve (AUC), 0.926; sensitivity, 90.0%; specificity, 86.7%, P<0.001] and IL-6 combined with hsa-miR-126-5p (AUC, 0.952; sensitivity, 93.3%; specificity, 90.0%; P<0.001) were able to better distinguish MAP from SAP than IL-1/IL-6 combined with hsa-miR-548d-5p, lipase, and amylase. IL-1 or IL-6 combined with hsa-miR-548d-5p (AUC, 0.924; sensitivity, 83.3%; specificity, 93.3%; P<0.001) were able to better distinguish SAP from RAP than IL-1/IL-6 combined with hsa-miR-126-5p, lipase, and amylase. IL-1 combined with hsa-miR-126-5p (AUC, 0.926; sensitivity, 90.0%; specificity, 86.7%; P<0.001) and IL-6 combined with hsa-miR-126-5p (AUC, 0.952; sensitivity, 93.3%; specificity, 90.0%; P<0.001) were able to better differentiate between MAP and RAP than IL-1/IL-6 combined with hsa-miR-548d-5p, lipase, and amylase. These results demonstrated that the combined detection of serum IL-6 and hsa-miR-126-5p may be useful for the early prediction of AP classification.

Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro.

The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20+ human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions.

The Metabolomic Study of Learning and Memory Function of APP/PS1 Double Transgenic Mice Improved by Gouteng San / 现代生物医学进展

Objective:The non targeted high-throughput urine metabolomics technology was used to study the pathogenesis of APP/PS 1 double transgenic mice and the mechanism of action of Gouteng san.Methods:5-month-old APP/PS 1 double transgenic mice were test with Morris water maze for spatial learning ability.Then we employed the non targeted high-throughput urine metabolomics technology to study the pathogenesis of APP/PS1 double transgenic mice based on the metabolic network.The focus investigation of the key pathways and the observation of the treatment by Morris water maze and metabolic level have been used after spatial learning ability damaged confirmed.Results:The comparison between APP/PS1 double transgenic mice and normal mice suggested that a significant longer was existed in former,which was call-back by Gouteng san.With the non targeted high-throughput urine metabolomics analysis and pathway focused analysis,we found certain signals from metabolic profiling,which was identified to be 6 biomarkers associated with learning and memory function by mass spectrometry analysis or authoritative database.Respectively,they were taurine,pteroylglutamic acid,neopterin,glutaurine,2-oxoglutarate and dihydroneopterin.They were mainly related to taurine metabolism and folate metabolism and represented an effective callback.Conclusion:Gouteng san possess a favorable effect on learning and memory ability of APP/PS1 double transgenic mice,6 biomarkers may be a potential target for the pathogenesis of APP/PSI double transgenic mice and provide experimental basis for the study of Gouteng san.

Study of Waternut Herb Extract on Aβ SAMP8 Damage of Mouse Hippocampal neurons and the Protective Effect of Metabolomic Study / 现代生物医学进展

Objective:To study the metabolic mechanism of protective effect of waternut herb extract on primary Aβ SAMP8 damage of mouse hippocampal neurons by metabolic footprinting.Methods:MTT assay was used to determine the proliferation of primary hippocampal neurons in SAMP8 mice with Aβ damage,the effect for the first time on the basis of metabolic footprinting evaluation waternut herb extract.Focus on key metabolic pathways and related metabolic targets,mechanism of primary Aβ SAMP8 damage of mouse hippocampal neurons and pathogenesis of watemut herb extract.Results:MTT assay was used to measure the rate of cell proliferation.The results showed that the cell viability of the primary hippocampal neurons was significantly decreased in the Aβ SAMP8 mice.The study found that metabolic footprinting,compared with littermate wild-type mice,neuronal cell metabolism Aβ SAMP8 damage of mouse anomalies mainly concentrated in the metabolism of folic acid and taufine metabolism associated with nerve cells,by high-throughput mass spectrometric analysis and literature database retrieval to determine the 3 differential metabolites,respectively is L-disodoum alanine (L-Cysteic acid),dihydrofolate (Dihydrofolate),acid (Chorismate),the branch of small molecule metabolites through extract intervention after Amakusa callback trend obviously.Conclusion:the therapeutic effect of watemut herb extract on Aβ SAMP8 damage of mouse primary hippocampal neurons to a certain extent,3 biomarkers of this discovery may be a potential target of Aβ SAMP8 damage of mouse primary hippocampal neurons in the pathogenesis of waternut herb extract,given after these markers were callback trend in different degree,suggesting that watemut herb extract could regulate metabolism related enzymes and metabolic pathways to protect the purpose,to provide the experimental basis for the treatment of Alzheimer's disease watemut herb extract.

Reduction of antibiotic resistance genes in municipal wastewater effluent by advanced oxidation processes.

This study investigated the reduction of antibiotic resistance genes (ARGs), intI1 and 16S rRNA genes, by advanced oxidation processes (AOPs), namely Fenton oxidation (Fe(2+)/H2O2) and UV/H2O2 process. The ARGs include sul1, tetX, and tetG from municipal wastewater effluent. The results indicated that the Fenton oxidation and UV/H2O2 process could reduce selected ARGs effectively. Oxidation by the Fenton process was slightly better than that of the UV/H2O2 method. Particularly, for the Fenton oxidation, under the optimal condition wherein Fe(2+)/H2O2 had a molar ratio of 0.1 and a H2O2 concentration of 0.01molL(-1) with a pH of 3.0 and reaction time of 2h, 2.58-3.79 logs of target genes were removed. Under the initial effluent pH condition (pH=7.0), the removal was 2.26-3.35 logs. For the UV/H2O2 process, when the pH was 3.5 with a H2O2 concentration of 0.01molL(-1) accompanied by 30min of UV irradiation, all ARGs could achieve a reduction of 2.8-3.5 logs, and 1.55-2.32 logs at a pH of 7.0. The Fenton oxidation and UV/H2O2 process followed the first-order reaction kinetic model. The removal of target genes was affected by many parameters, including initial Fe(2+)/H2O2 molar ratios, H2O2 concentration, solution pH, and reaction time. Among these factors, reagent concentrations and pH values are the most important factors during AOPs.

Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins.

Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs.

Inactivation of antibiotic resistance genes in municipal wastewater effluent by chlorination and sequential UV/chlorination disinfection.

This study investigated disinfection methods including chlorination, ultraviolet (UV) irradiation and sequential UV/chlorination treatment on the inactivation of antibiotic resistance genes (ARGs). ARGs including sul1, tetX, tetG, intI1, and 16S rRNA genes in municipal wastewater treatment plant (MWTP) effluent were examined. The results indicated a positive correlation between the removal of ARGs and chlorine dosage (p=0.007-0.014, n=6),as well as contact time (p=0.0001, n=10). Greater free chlorine (FC) dosage leads to higher removal for all the genes and the maximum removal (1.30-1.49 logs) could be achieved at FC dosage of 30 mg L(-1). The transformation kinetic data for ARGs removal (log C0/C) followed the second-order reaction kinetic model with FC dosage (R(2)=0.6829-0.9999) and contact time (R(2)=0.7353-8634), respectively. Higher ammonia nitrogen (NH3-N) concentration was found to lead to lower removal of ARGs at the same chlorine dosage. When the applied Cl2:NH3-N ratio was over 7.6:1, a significant reduction of ARGs (1.20-1.49 logs) was achieved. By using single UV irradiation, the log removal values of tetX and 16Ss rRNA genes were 0.58 and 0.60, respectively, while other genes were 0.36-0.40 at a fluence of 249.5 mJ cm(-2), which was observed to be less effective than chlorination. With sequential UV/chlorination treatment, 0.006 to 0.31 log synergy values of target genes were observed under different operation parameters.

A multifactorial screening strategy to identify anti-idiotypic reagents for bioanalytical support of antibody therapeutics.

Antibodies are critical tools for protein bioanalysis; their quality and performance dictate the caliber and robustness of ligand binding assays. After immunization, polyclonal B cells generate a diverse antibody repertoire against constant and variable regions of the therapeutic antibody immunogen. Herein we describe a comprehensive and multifactorial screening strategy to eliminate undesirable constant region-specific antibodies and select for anti-idiotypic antibodies with specificity for the unique variable region. Application of this strategy is described for the therapeutic antibody Mab-A case study. Five different factors were evaluated to select a final antibody pair for the quantification of therapeutics in biological matrices: (i) matrix effect in preclinical and clinical matrices, (ii) assay sensitivity with lower limit of quantification goal of single-digit ng/ml (low pM) at a signal-to-background ratio greater than 5, (iii) epitope distinction or nonbridging antibody pair, (iv) competition with target and inhibitory capacity enabling measurement of free drug, and (v) neutralizing bioactivity using bioassay. The selected antibody pair demonstrated superior assay sensitivity with no or minimal matrix effect in common biological samples, recognized two distinct binding epitopes on the therapeutic antibody variable region, and featured inhibitory and neutralizing effects with respect to quantification of free drug levels.