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Biomedicines May 2024The CRISPR-Cas9 system is a revolutionary tool in genetic engineering, offering unprecedented precision and efficiency in genome editing. Cas9, an enzyme derived from...
The CRISPR-Cas9 system is a revolutionary tool in genetic engineering, offering unprecedented precision and efficiency in genome editing. Cas9, an enzyme derived from bacteria, is guided by RNA to edit DNA sequences within cells precisely. However, while CRISPR-Cas9 presents notable benefits and encouraging outcomes as a molecular tool and a potential therapeutic agent, the process of producing and purifying recombinant Cas9 protein remains a formidable hurdle. In this study, we systematically investigated the expression of recombinant SpCas9-His in four distinct () strains (Rosetta2, BL21(DE3), BL21(DE3)-pLysS, and BL21(DE3)-Star). Through optimization of culture conditions, including temperature and post-induction time, the BL21(DE3)-pLysS strain demonstrated efficient SpCas9 protein expression. This study also presents a detailed protocol for the purification of recombinant SpCas9, along with detailed troubleshooting tips. Results indicate successful SpCas9 protein expression using BL21(DE3)-pLysS at 0.5 mM IPTG concentration. Furthermore, the findings suggest potential avenues for further enhancements, paving the way for large-scale Cas9 production. This research contributes valuable insights into optimizing strains and culture conditions for enhanced Cas9 expression, offering a step forward in the development of efficient genome editing tools and therapeutic proteins.
PubMed: 38927433
DOI: 10.3390/biomedicines12061226 -
Antibiotics (Basel, Switzerland) Jun 2024The emergence of as a multidrug-resistant fungal pathogen represents a significant global health challenge, especially given the growing issue of antifungal drug... (Review)
Review
The emergence of as a multidrug-resistant fungal pathogen represents a significant global health challenge, especially given the growing issue of antifungal drug resistance. This review aims to illuminate the potential of essential oils (EOs), which are volatile plant secretions containing complex mixtures of chemicals, as alternative antifungal agents to combat , thus combining traditional insights with contemporary scientific findings to address this critical health issue. A systematic literature review was conducted using the PubMed, Scopus, and Web of Science databases from 2019 to 2024, and using the Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) protocol to identify relevant studies on the antifungal efficacy of EOs or their components against . Of the 90 articles identified, 16 were selected for detailed review. The findings highlight the diverse mechanisms of action of EOs and their components, such as disrupting fungal cell membranes, inducing the production of reactive oxygen species (ROS), and impeding biofilm formation, suggesting that some of them may be as effective as, or better than, traditional antifungal drugs while potentially limiting the development of resistance. However, issues such as variability in the composition of EOs and a paucity of clinical trials have been identified as significant obstacles. In conclusion, EOs and their active ingredients are emerging as viable candidates for creating effective treatments for , underscoring their importance as alternative or complementary antifungal agents in the face of increasing drug resistance. The call for future research underscores the need for clinical trials and standardization to unlock the full antifungal potential of EOs against .
PubMed: 38927234
DOI: 10.3390/antibiotics13060568 -
Antibiotics (Basel, Switzerland) Jun 2024Bloodstream infections (BSI) caused by multidrug-resistant (MDR) bacteria, pose a major threat for patients, especially for those who are immunosuppressed. Rapid...
Bloodstream infections (BSI) caused by multidrug-resistant (MDR) bacteria, pose a major threat for patients, especially for those who are immunosuppressed. Rapid pathogen detection and characterization from positive blood cultures are crucial in the management of patients with BSI to enable an adequate and timely antimicrobial therapy. This study aimed to investigate the potential role of the Molecular Mouse system, a new CE IVD molecular test designed to rapidly detect the causative agents of bacteremia and their resistance determinants, in the management of the therapy in critically ill patients. Agreement between the results of the Molecular Mouse and the conventional routine method was also considered. Overall, 100 positive blood cultures were collected from septic critically ill patients from May 2023 to January 2024 and analyzed with Molecular Mouse and routine protocols. The new instrument consistently agreed with the routine protocols in the case of monomicrobial blood cultures, while some discrepancies were obtained in the polymicrobial samples. Antimicrobial resistance genes were detected in 35 samples, with and CTX-M-1/9 groups being the most frequently detected targets. Therapy was adjusted in 42 critically ill patients confirming the importance of new rapid molecular tests in the management of positive blood cultures, to adjust empirical therapy and use new antibiotics accurately.
PubMed: 38927183
DOI: 10.3390/antibiotics13060517 -
Scientific Reports Jun 2024Idiopathic Pulmonary Fibrosis (IPF) is a debilitating and fatal lung disease characterized by the excessive formation of scar tissue and decline of lung function....
Idiopathic Pulmonary Fibrosis (IPF) is a debilitating and fatal lung disease characterized by the excessive formation of scar tissue and decline of lung function. Despite extensive research, only two FDA-approved drugs exist for IPF, with limited efficacy and relevant side effects. Thus, there is an urgent need for new effective therapies, whose discovery strongly relies on IPF animal models. Despite some limitations, the Bleomycin (BLM)-induced lung fibrosis mouse model is widely used for antifibrotic drug discovery and for investigating disease pathogenesis. The initial acute inflammation triggered by BLM instillation and the spontaneous fibrosis resolution that occurs after 3 weeks are the major drawbacks of this system. In the present study, we applied micro-CT technology to a longer-lasting, triple BLM administration fibrosis mouse model to define the best time-window for Nintedanib (NINT) treatment. Two different treatment regimens were examined, with a daily NINT administration from day 7 to 28 (NINT 7-28), and from day 14 to 28 (NINT 14-28). For the first time, we automatically derived both morphological and functional readouts from longitudinal micro-CT. NINT 14-28 showed significant effects on morphological parameters after just 1 week of treatment, while no modulations of these biomarkers were observed during the preceding 7-14-days period, likely due to persistent inflammation. Micro-CT morphological data evaluated on day 28 were confirmed by lung histology and bronchoalveolar lavage fluid (BALF) cells; Once again, the NINT 7-21 regimen did not provide substantial benefits over the NINT 14-28. Interestingly, both NINT treatments failed to improve micro-CT-derived functional parameters. Altogether, our findings support the need for optimized protocols in preclinical studies to expedite the drug discovery process for antifibrotic agents. This study represents a significant advancement in pulmonary fibrosis animal modeling and antifibrotic treatment understanding, with the potential for improved translatability through the concurrent structural-functional analysis offered by longitudinal micro-CT.
Topics: Animals; Bleomycin; Disease Models, Animal; Mice; X-Ray Microtomography; Indoles; Antifibrotic Agents; Lung; Pulmonary Fibrosis; Idiopathic Pulmonary Fibrosis; Mice, Inbred C57BL; Time Factors
PubMed: 38926490
DOI: 10.1038/s41598-024-65030-3 -
BMJ Open Jun 2024Norepinephrine (NE) is the first-line recommended vasopressor for restoring mean arterial pressure (MAP) in vasoplegic syndrome (vs) following cardiac surgery with...
Norepinephrine weaning guided by the Hypotension Prediction Index in vasoplegic shock after cardiac surgery: protocol for a single-centre, open-label randomised controlled trial - the NORAHPI study.
INTRODUCTION
Norepinephrine (NE) is the first-line recommended vasopressor for restoring mean arterial pressure (MAP) in vasoplegic syndrome (vs) following cardiac surgery with cardiopulmonary bypass. However, solely focusing on target MAP values can lead to acute hypotension episodes during NE weaning. The Hypotension Prediction Index (HPI) is a machine learning algorithm embedded in the Acumen IQ device, capable of detecting hypotensive episodes before their clinical manifestation. This study evaluates the clinical benefits of an NE weaning strategy guided by the HPI.
MATERIAL AND ANALYSIS
The Norahpi trial is a prospective, open-label, single-centre study that randomises 142 patients. Inclusion criteria encompass adult patients scheduled for on-pump cardiac surgery with postsurgical NE administration for vs patient randomisation occurs once they achieve haemodynamic stability (MAP>65 mm Hg) for at least 4 hours on NE. Patients will be allocated to the intervention group (n=71) or the control group (n=71). In the intervention group, the NE weaning protocol is based on MAP>65 mmHg and HPI<80 and solely on MAP>65 mm Hg in the control group. Successful NE weaning is defined as achieving NE weaning within 72 hours of inclusion. An intention-to-treat analysis will be performed. The primary endpoint will compare the duration of NE administration between the two groups. The secondary endpoints will include the prevalence, frequency and time of arterial hypotensive events monitored by the Acumen IQ device. Additionally, we will assess cumulative diuresis, the total dose of NE, and the number of protocol weaning failures. We also aim to evaluate the occurrence of postoperative complications, the length of stay and all-cause mortality at 30 days.
ETHICS AND DISSEMINATION
Ethical approval has been secured from the Institutional Review Board (IRB) at the University Hospital of Amiens (IRB-ID:2023-A01058-37). The findings will be shared through peer-reviewed publications and presentations at national and international conferences.
TRIAL REGISTRATION NUMBER
NCT05922982.
Topics: Humans; Vasoplegia; Hypotension; Prospective Studies; Norepinephrine; Cardiac Surgical Procedures; Vasoconstrictor Agents; Randomized Controlled Trials as Topic; Postoperative Complications; Machine Learning
PubMed: 38926148
DOI: 10.1136/bmjopen-2024-084499 -
Discovery Medicine Jun 2024Atypical acinar cell foci (AACF) seen in pancreatic cancer are fatal and have been studied with some causative agents. However, for the first time, the effect of...
BACKGROUND
Atypical acinar cell foci (AACF) seen in pancreatic cancer are fatal and have been studied with some causative agents. However, for the first time, the effect of acetylsalicylic acid with nitric oxide (NO-ASA) on AACF was examined in this study. Although NO-ASA has very successful inhibitory effects against some types of cancer, it has not been investigated whether they can exert their inhibition effects on AACFs.
METHODS
For experimental purposes, 21 14-day-old male Wistar albino rats were used. Azaserine (30 mg/kg) was dissolved in 0.9% NaCl solution and injected intraperitoneally (i.p.) into 14 rats, except for the Control group (Cont) rats, for three weeks. Rats that were injected with azaserine once a week for three weeks and those that did not receive treatment were divided into experimental groups. 15 days after the end of the azaserine injection protocol, NO-ASA was applied to azaserine with NO-ASA (Az+NO-ASA) group rats three consecutive times with an interval of 15 days by gavage. At the end of the 5-month period, pancreatic tissue was dissected and weighed. Pancreas preparations prepared from histological sections were examined for AACF burden and analyzed via a video image analyzer. One-way analysis of variance (ANOVA) non-parametric statistical analyses were performed to test whether there was a difference between the averages of the experimental and Control groups.
RESULTS
AACF burden in both groups injected with azaserine was found to be statistically significant in all categories compared to that of the Control group ( < 0.05). The average Calculated Estimated average AACF volume (mm) values, the Calculated estimated average AACF diameter (μm), the Estimated average number of AACF per unit volume, AACF rate as a % of Calculated Organ Volume were higher in the AzCont group rats than in the Az+NO-ASA group, when compared, and there was an important level statistical difference between the groups ( < 0.05). It was determined that for all parameters AACFs load in Az+NO-ASA group rats were significantly reduced compared to that of AzCont group rats ( < 0.05).
CONCLUSIONS
We observed that, as a result of the NO-ASA application, the experimental AACF focus ratio created by azaserine injection was significantly inhibited. The inhibitory effect of AACFs in Az+NO-ASA group rats may have resulted from the significant and independent chemopreventive and/or chemotherapeutic activity of NO-ASA against exocrine pancreatic AACF foci.
Topics: Animals; Male; Aspirin; Rats, Wistar; Nitric Oxide; Rats; Pancreatic Neoplasms; Acinar Cells; Pancreas, Exocrine
PubMed: 38926102
DOI: 10.24976/Discov.Med.202436185.106 -
Heart (British Cardiac Society) Jun 2024Despite restoration of epicardial blood flow in acute ST-elevation myocardial infarction (STEMI), inadequate microcirculatory perfusion is common and portends a poor...
BACKGROUND
Despite restoration of epicardial blood flow in acute ST-elevation myocardial infarction (STEMI), inadequate microcirculatory perfusion is common and portends a poor prognosis. Intracoronary (IC) thrombolytic therapy can reduce microvascular thrombotic burden; however, contemporary studies have produced conflicting outcomes.
OBJECTIVES
This meta-analysis aims to evaluate the efficacy and safety of adjunctive IC thrombolytic therapy at the time of primary percutaneous coronary intervention (PCI) among patients with STEMI.
METHODS
Comprehensive literature search of six electronic databases identified relevant randomised controlled trials. The primary outcome was major adverse cardiac events (MACE). The pooled risk ratio (RR) and weighted mean difference (WMD) with a 95% CI were calculated.
RESULTS
12 studies with 1915 patients were included. IC thrombolysis was associated with a significantly lower incidence of MACE (RR=0.65, 95% CI 0.51 to 0.82, I=0%, p<0.0004) and improved left ventricular ejection fraction (WMD=1.87; 95% CI 1.07 to 2.67; I=25%; p<0.0001). Subgroup analysis demonstrated a significant reduction in MACE for trials using non-fibrin (RR=0.39, 95% CI 0.20 to 0.78, I=0%, p=0.007) and moderately fibrin-specific thrombolytic agents (RR=0.62, 95% CI 0.47 to 0.83, I=0%, p=0.001). No significant reduction was observed in studies using highly fibrin-specific thrombolytic agents (RR=1.10, 95% CI 0.62 to 1.96, I=0%, p=0.75). Furthermore, there were no significant differences in mortality (RR=0.91; 95% CI 0.48 to 1.71; I=0%; p=0.77) or bleeding events (major bleeding, RR=1.24; 95% CI 0.47 to 3.28; I=0%; p=0.67; minor bleeding, RR=1.47; 95% CI 0.90 to 2.40; I=0%; p=0.12).
CONCLUSION
Adjunctive IC thrombolysis at the time of primary PCI in patients with STEMI improves clinical and myocardial perfusion parameters without an increased rate of bleeding. Further research is needed to optimise the selection of thrombolytic agents and treatment protocols.
PubMed: 38925881
DOI: 10.1136/heartjnl-2024-324078 -
Anticancer Research Jul 2024Sorafenib and lenvatinib have long been used as a first-line treatment for advanced hepatocellular carcinoma (HCC). Along with the development of systemic chemotherapy...
BACKGROUND/AIM
Sorafenib and lenvatinib have long been used as a first-line treatment for advanced hepatocellular carcinoma (HCC). Along with the development of systemic chemotherapy for HCC, the concept of conversion hepatectomy has recently become widespread. The present study aimed to assess the clinical outcomes of sorafenib and lenvatinib for HCC regarding the possibility of conversion hepatectomy in clinical practice.
PATIENTS AND METHODS
A total of 295 patients with advanced HCC receiving sorafenib and lenvatinib, accounting for 306 treatments (sorafenib, n=157; lenvatinib, n=149, 11 patients received lenvatinib after sorafenib treatment) at five different institutions were enrolled. Patients were assessed for their clinical characteristics and therapeutic response using both Response Evaluation Criteria in Solid Tumors criteria (RECIST) and modified RECIST (mRECIST) criteria. Additionally, an indication of surgery after tyrosine kinase inhibitor administration was determined based on the tumor status of patients.
RESULTS
The median survival times of patients treated with sorafenib and lenvatinib were 12.8 and 16.4 months, respectively, without significant difference (p=0.1645). The objective response rates (ORR) of sorafenib based on mRECIST and RECIST were 10.1% and 5.9%, respectively, and those of lenvatinib were 38.1% and 19.0%, respectively. Among the 306 treatments, two cases (sorafenib and lenvatinib, one each) underwent hepatectomy after systemic chemotherapy.
CONCLUSION
Few cases with unresectable HCC were amenable to conversion hepatectomy after sorafenib and lenvatinib treatments due to the limited ORR by RECIST. Cautious approach must be taken when administering neoadjuvant chemotherapy aimed at conversion hepatectomy.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; Quinolines; Phenylurea Compounds; Sorafenib; Male; Female; Hepatectomy; Middle Aged; Aged; Adult; Aged, 80 and over; Treatment Outcome; Antineoplastic Combined Chemotherapy Protocols; Antineoplastic Agents
PubMed: 38925835
DOI: 10.21873/anticanres.17123 -
Anticancer Research Jul 2024The therapeutic landscape for metastatic castration-resistant prostate cancer (mCRPC) has changed dramatically with the introduction of several novel agents. However,...
BACKGROUND/AIM
The therapeutic landscape for metastatic castration-resistant prostate cancer (mCRPC) has changed dramatically with the introduction of several novel agents. However, limited data are available to determine whether the introduction of novel agents affected patient characteristics, treatment patterns, and outcomes compared with the period when these agents were not available. The objective of this study was to evaluate the impact of the introduction of novel mCRPC agents on patient characteristics, treatment patterns, and outcomes.
PATIENTS AND METHODS
Two cohorts of Japanese patients diagnosed with mCRPC between 2009 and 2014 (Epoch 1) and 2015 and 2019 (Epoch 2) were retrospectively analyzed.
RESULTS
A total of 125 treatment-naïve mCRPC patients, consisting of 42 patients in Epoch 1 and 83 patients in Epoch 2, were evaluated. We obtained the following results: (i) a dramatic shift in the first-line treatment from docetaxel to androgen receptor axis-targeted agents (ARATs), (ii) an age range expansion for first-line treatment, and (iii) an overall survival (OS) advantage in Eopch 2 compared to Epoch 1. Multivariate analysis in the overall population showed that Epoch 2 and low prostate-specific antigen (PSA) levels at the start of first-line treatment were independent prognostic factors for OS.
CONCLUSION
In real-world mCRPC practice, the introduction of novel agents has improved the prognosis of mCRPC while allowing more patients to receive mCRPC treatment across a broad age range. In addition, low PSA levels at the start of first-line treatment were associated with improved OS, indicating the importance of starting mCRPC treatment while PSA levels are low.
Topics: Humans; Male; Prostatic Neoplasms, Castration-Resistant; Aged; Aged, 80 and over; Retrospective Studies; Middle Aged; Treatment Outcome; Docetaxel; Prostate-Specific Antigen; Neoplasm Metastasis; Antineoplastic Combined Chemotherapy Protocols; Antineoplastic Agents
PubMed: 38925833
DOI: 10.21873/anticanres.17130 -
Anticancer Research Jul 2024Randomized trials have shown the benefit of combining tyrosine kinase inhibitors (TKI) and chemotherapy in the treatment of epidermal growth factor receptor-mutant...
BACKGROUND/AIM
Randomized trials have shown the benefit of combining tyrosine kinase inhibitors (TKI) and chemotherapy in the treatment of epidermal growth factor receptor-mutant non-small-cell lung cancer (NSCLC). For anaplastic lymphoma kinase-rearranged (ALK) NSCLC, prospective trial results of the combination are not available and have not even been thoroughly investigated in vitro. In this study, we investigated combinations of TKI and chemotherapy using in vitro models of ALK NSCLC.
MATERIALS AND METHODS
ALK cell line models H3122, H2228, and DFCI032 with differing primary resistance to ALK receptor TKIs were used. We investigated short-(viability assay) and long-term (colony-formation assay) cytotoxicity, apoptosis, and cell signaling in response to the combinations of agents. We selected the most commonly used agents, alectinib, cisplatin, and pemetrexed, to investigate the combination effects.
RESULTS
In the combination experiments with short-term exposure, synergism between TKI and pemetrexed was observed, while cisplatin had antagonistic effects. In the long-term experiments, the combination of cisplatin and TKI was synergistic in all lines, while no synergism was observed with pemetrexed. Among the chemotherapy and TKI sequences, cisplatin followed by TKI was more cytotoxic than the opposite in two out of the three models. In the TKI-sensitive H3122 cell line, the combination of chemotherapy and TKI combination increased apoptosis. Interestingly, pemetrexed treatment resulted in the activation of ALK, which was abolished with TKI.
CONCLUSION
Combining TKI and chemotherapy in ALK models has some synergistic effects that overcome primary TKI resistance. However, the synergy varies depending on the chemotherapeutic agent, cytotoxic assay, and the cell line used. Prospective clinical trials are warranted to fully characterize the potential of combination chemotherapy with TKIs in ALK NSCLC.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Anaplastic Lymphoma Kinase; Lung Neoplasms; Protein Kinase Inhibitors; Cell Line, Tumor; Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Pemetrexed; Apoptosis; Drug Synergism; Drug Resistance, Neoplasm; Receptor Protein-Tyrosine Kinases; Piperidines; Carbazoles
PubMed: 38925827
DOI: 10.21873/anticanres.17092