tRNA engineering strategies for genetic code expansion.

The advancement of genetic code expansion (GCE) technology is attributed to the establishment of specific aminoacyl-tRNA synthetase/tRNA pairs. While earlier improvements mainly focused on aminoacyl-tRNA synthetases, recent studies have highlighted the importance of optimizing tRNA sequences to enhance both unnatural amino acid incorporation efficiency and orthogonality. Given the crucial role of tRNAs in the translation process and their substantial impact on overall GCE efficiency, ongoing efforts are dedicated to the development of tRNA engineering techniques. This review explores diverse tRNA engineering approaches and provides illustrative examples in the context of GCE, offering insights into the user-friendly implementation of GCE technology.

Automated sepsis detection with vancomycin- and allantoin-polydopamine magnetic nanoparticles.

Rapid and accurate identification of the bacteria responsible for sepsis is paramount for effective patient care. Molecular diagnostic methods, such as polymerase chain reaction (PCR), encounter challenges in sepsis due to inhibitory compounds in the blood, necessitating their removal for precise analysis. In this study we present an innovative approach that utilizes vancomycin (Van) and allantoin (Al)-conjugated polydopamine (PDA)-coated magnetic nanoparticles (MNPs) for the rapid and automated enrichment of bacteria and their DNA extraction from blood without inducing clumping and aggregation of blood. Al/Van-PDA-MNPs, facilitated by IMS, eliminate the need for preliminary sample treatments, providing a swift and efficient method for bacterial concentration and DNA extraction within an hour. Employing Al/Van-PDA-MNPs within an automated framework has markedly improved our ability to pre-concentrate various Gram-negative and Gram-positive bacteria directly from blood samples. This advancement has effectively reduced the detection threshold to 102 colony-forming unit/mL by both PCR and quantitative PCR. The method's expedited processing time, combined with its precision, positions it as a feasible diagnostic tool for diverse healthcare settings, ranging from small clinics to large hospitals. Furthermore, the innovative application of nanoparticles for DNA extraction holds promising potential for advancing sepsis diagnostics, enabling earlier interventions and improving patient outcomes.

3D printed fluidic swab for COVID-19 testing with improved diagnostic yield and user comfort.

The current standard method of diagnosing coronavirus disease 2019 (COVID-19) involves uncomfortable and invasive nasopharyngeal (NP) sampling using cotton swabs (CS), which can be unsuitable for self-testing. Although mid-turbinate sampling is an alternative, it has a lower diagnostic yield than NP sampling. Nasal wash (NW) has a similar diagnostic yield to NP sampling, but is cumbersome to perform. In this study, we introduce a 3D printed fluidic swab (3DPFS) that enables easy NW sampling for COVID-19 testing with improved diagnostic yield. The 3DPFS comprises a swab head, microchannel, and socket that can be connected to a syringe containing 250 µL of NW solution. The 3DPFS efficiently collects nasal fluid from the surface of the nasal cavity, resulting in higher sensitivity than CS for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This was confirmed by both reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and lateral flow assays (LFA) in virus-spiked nasal samples and clinical samples. Additionally, users reported greater comfort when using the 3DPFS compared to CS. These findings suggest that the 3DPFS can improve the performance of COVID-19 testing by facilitating efficient and less painful nasal sample collection.

Bioorthogonal CRISPR/Cas9-Drug Conjugate: A Combinatorial Nanomedicine Platform.

Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.

A Versatile Strategy for Screening Custom-Designed Warhead-Armed Cyclic Peptide Inhibitors.

Demand for peptide-based pharmaceuticals has been steadily increasing, but only limited success has been achieved to date. To expedite peptide-based drug discovery, we developed a general scheme for cell-based screening of cyclic peptide inhibitors armed with a user-designed warhead. We combined unnatural amino acid incorporation and split intein-mediated peptide cyclization techniques and integrated a yeast-based colorimetric screening assay to generate a new scheme that we call the custom-designed warhead-armed cyclic peptide screening platform (CWCPS). This strategy successfully discovered a potent inhibitor, CY5-6Q, that targets human histone deacetylase 8 (HDAC8) with a KD value of 15 nM. This approach can be a versatile and general platform for discovering cyclic peptide inhibitors.

The trend of ammonia levels in patients with glufosinate ammonium poisoning with respect to neurotoxicity.

Since glufosinate irreversibly inhibits glutamine synthetase, leading to intracellular accumulation of ammonia, hyperammonemia is considered one of the main mechanisms of glufosinate ammonium toxicity in humans. However, whether hyperammonemia causes neurotoxicity has not yet been studied. Therefore, the purpose of this study was to determine whether the serum ammonia level is elevated before the development of neurotoxicity. In this retrospective observational study, we analyzed data from consecutive patients diagnosed with acute glufosinate ammonium poisoning. The primary outcome was the development of neurotoxicity following the poisoning. Patients who developed neurotoxicity were characterized by higher initial ammonia levels compared to patients without neurotoxicity (121.0 µg/dL [87.0; 141.0] vs 83.0 µg/dL [65.0; 119.0], p < 0.01). However, there was no increase in ammonia levels over time in both the asymptomatic and neurotoxicity groups when serial serum ammonia levels were examined from emergency department admission to hospital discharge. In addition, there was no statistically significant difference between the peak ammonia levels in the asymptomatic group and the peak ammonia levels before symptom onset in the neurotoxicity group (135.0 µg/dL [109.0; 158.0] vs 144.0 µg/dL [120.0; 189.0], p = 0.15). Following the onset of neurotoxicity, the serum ammonia level increased significantly (125.0 [111.0; 151.0] µg/dL to 148.0 [118.0; 183.0] µg/dL, p < 0.01). In conclusion, hyperammonemia cannot be assumed as the cause of neurotoxicity in glufosinate ammonium poisoning and further research is needed to examine the exact mechanism of GA poisoning.

Prognostic value of neutrophil to lymphocyte ratio in the diagnosis of neurotoxicity after glufosinate ammonium poisoning.

Neurotoxicity related to glufosinate ammonium is known to occur after a latent period of 4-60 hr following ingestion of this herbicide. However, neurotoxicity is difficult to predict in the emergency department (ED) and only a few parameters are known to be useful to indicate development of neurotoxicity. Determination of a systemic inflammation parameter such as the neutrophil to lymphocyte ratio (NLR), is a rapid and simple method which was found to be a prognostic marker in various clinical conditions such as sepsis, cardiac disorders, stroke, and cancer. Therefore, the aim of this study was to determine whether the NLR might predict neurotoxicity and be used at ED to detect neurotoxicity induced following glufosinate ammonium poisoning in admitted patients. This retrospective observational study collected data from consecutive patients diagnosed with acute glufosinate ammonium poisoning between January 2005 and December 2020. The primary outcome was development of neurotoxicity following acute glufosinate ammonium poisoning. Out of the 72 patients selected 44 patients (61.1%) exhibited neurotoxic symptoms. Neurotoxicity appeared with an approximate latent period of 12 hr. The NLR was significantly higher in the group displaying neurotoxicity. Multivariable analysis showed that the NLR was significant in predicting neurotoxicity. The NLR was independently associated with neurotoxicity initiated by glufosinate ammonium. Therefore, the use of the NLR might help clinically to readily and rapidly predict development of neurotoxicity associated with glufosinate ammonium at the ED.

Diagnostic value of transthoracic echocardiography compared to electrocardiogram in predicting coronary artery stenosis among patients after cardiac arrest.

BACKGROUND: In the absence of ST-segment elevation (STE) in post-return of spontaneous circulation (ROSC) electrocardiogram (ECG), coronary angiography (CAG) is required in patients with suspected coronary artery disease (CAD). However, it is a challenge to identify patients with CAD after cardiac arrest (CA). Recent European Society of Cardiology guidelines recommends transthoracic echocardiography in patients presenting with cardiac arrest. We aimed to assess the diagnostic value of regional wall motion abnormalities (RWMAs) on transthoracic echocardiography (TTE) compared to ECG in diagnosing significant coronary artery stenosis in CA patients. METHODS: This is a retrospective, observational study of adult CA patients with presumed cardiac etiology who underwent CAG from a single tertiary care hospital. We compared the predictive value of RWMA on TTE and STE on ECG in significant stenosis of ≥70% of the coronary artery diameter. The primary outcome was significant stenosis on CAG. RESULTS: There were 145 patients included in this study and CAG revealed significant stenosis in 76 (52.4%) patients. Among the 76 patients with significant stenosis, 68 (89.5%) had RWMA on TTE and 41 (54.0%) had STE. RWMA on TTE (OR 3.67; 95% CI 1.52-8.85) was independently associated with significant stenosis. Combining both RWMA on TTE and STE on ECG improved performance in the receiver operating characteristic curve analysis (area under the curve 0.722) for predicting significant stenosis compared to using only ECG alone (p = 0.001). CONCLUSIONS: RWMAs on TTE was independently associated with significant stenosis. The RWMA and STE combination had better predictive performance than using only STE on ECG to predict significant stenosis.

Is two-dimensional echocardiography better than electrocardiography for predicting patient outcomes after cardiac arrest?

BACKGROUND: Coronary artery stenosis increases hospital mortality and leads to poor neurological recovery in cardiac arrest (CA) patients. However, electrocardiography (ECG) cannot fully predict the presence of coronary artery stenosis in CA patients. Hence, we aimed to determine whether regional wall motion abnormality (RWMA), as observed by two-dimensional echocardiography (2DE), predicted patient survival outcomes with greater accuracy than did ST segment elevation (STE) on ECG in CA patients who underwent coronary angiography (CAG) after return of spontaneous circulation. METHODS: This was a retrospective observational study of adult patients with CA of presumed cardiac etiology who underwent CAG at a single tertiary care hospital. We investigated whether RWMA observed on 2DE predicted patient outcomes more accurately than did STE observed on ECG. The primary outcome was incidence of hospital mortality. The secondary outcomes were Glasgow-Pittsburgh Cerebral Performance Category scores measured 6 months after discharge and significant coronary artery stenosis on CAG. RESULTS: Among the 145 patients, 36 (24.8%) experienced in-hospital death. In multivariable analysis of survival outcomes, only total arrest time (P=0.011) and STE (P=0.035) were significant. The odds ratio (OR) and 95% confidence interval (CI), which were obtained by adjusting the total arrest time for survival outcomes, were significant only for STE (OR, 0.40; 95% CI, 0.17-0.94). The presence of RWMA was not a significant factor. CONCLUSIONS: While STE predicted survival outcomes in adult CA patients, RWMA did not. The decision to perform CAG after CA should include ECG under existing guidelines. The use of RWMA has limited benefits in treatment of this population.