Chemical optimization and derivatization of micrococcin p2 to target multiple bacterial infections: new antibiotics from thiopeptides.

Antimicrobial resistance poses a significant threat to humanity, and the development of new antibiotics is urgently needed. Our research has focused on thiopeptide antibiotics such as micrococcin P2 (MP2) and derivatives thereof as new anti-infective agents. Thiopeptides are sulfur-rich, structurally complex substances that exhibit potent activity against Gram-positive pathogens and Mycobacteria species, including clinically resistant strains. The clinical development of thiopeptides has been hampered by the lack of efficient synthetic platforms to conduct detailed structure-activity relationship studies of these natural products. The present contribution touches upon efficient synthetic routes to MP2 that laid the groundwork for clinical translation. The medicinal chemistry campaign on MP2 has been guided by computational molecular dynamic simulations and parallel investigations to improve drug-like properties, such as enhancing the aqueous solubility and optimizing antibacterial activity. Such endeavors have enabled identification of promising lead compounds, AJ-037 and AJ-206, against Mycobacterium avium complex (MAC). Extensive in vitro studies revealed that these compounds exert potent activity against MAC species, a subspecies of non-tuberculous mycobacteria (NTM) that proliferate inside macrophages. Two additional pre-clinical candidates have been identified: AJ-024, for the treatment of Clostridioides difficile infections, and AJ-147, for methicillin-resistant Staphylococcus aureus impetigo. Both compounds compare quite favorably with current first-line treatments. In particular, the ability of AJ-147 to downregulate pro-inflammatory cytokines adds a valuable dimension to its clinical use. In light of above, these new thiopeptide derivatives are well-poised for further clinical development.

Monomer-mixed hole transport layers for improving hole injection of quantum dot light-emitting diodes.

Quantum-dot light-emitting diodes (QLEDs) are promising components for next-generation displays and related applications. However, their performance is critically limited by inherent hole-injection barrier caused by deep highest-occupied molecular orbital levels of quantum dots. Herein, we present an effective method for enhancing the performance of QLEDs by incorporating a monomer (TCTA or mCP) into hole-transport layers (HTL). The impact of different monomer concentrations on the characteristics of QLEDs were investigated. The results indicate that sufficient monomer concentrations improve the current efficiency and power efficiency. The increased hole current using monomer-mixed HTL suggests that our method holds considerable potential for high-performance QLEDs.

Difference in Sugar-Sweetened Beverage Consumption before and during the COVID-19 Pandemic among Korean Adolescents.

BACKGROUND: Previous studies have identified various factors associated with sugar-sweetened beverage (SSB) consumption among children and adolescents. Recent studies attempted to analyze changes in SSB consumption of adolescents during the COVID-19 pandemic and showed conflicting results. OBJECTIVES: This study aimed to estimate the difference in SSB consumption before (2018-2019) and during (2020-2021) the COVID-19 pandemic among Korean adolescents. METHODS: The study population consisted of students (n = 227,139) aged 12-18 y from the Korean Youth Risk Behavior Web-based Survey (KYRBWS). Data collection was done between 2018 and 2021. The primary outcome was the difference in the SSB consumption status (none/<7 times/wk, ≥7 times/wk) before and during the COVID-19 pandemic. Multinomial logistic regression was used to examine the association. Additional analysises were also conducted by gender, school grades, household income, grade point average, region, household members, fast-food intake, and fruit intake. RESULTS: The COVID-19 pandemic was associated with a decrease in adolescents' SSB intake. [(<7 times/wk) 2019: 59.4, 2020: 58.8, (≥7 times/wk) 2019: 35.3, 2020: 33.4]. CONCLUSIONS: The study found a difference in SSB consumption among Korean adolescents between before and during the COVID-19 pandemic. These findings are noteworthy considering the importance of continuous care in managing SSB intake.

Did the increase in sitting time due to COVID-19 lead to obesity in adolescents?

INTRODUCTION: Since adolescent with obesity is closely linked with the incidence of cardiovascular disease, it is important to identify the factors that increase the prevalence of adolescent with obesity and prevent it early. This study aimed to examine which of the demographic and lifestyle factors including sitting hours per week for purposes other than study had the greatest influence on Korean adolescents with obesity during the coronavirus disease 2019 (COVID-19) pandemic. METHODS: We used the Korean Youth Risk Behavior Web-based Survey (KYRBWS) data. The primary outcome was the relationship between sitting hours and obesity during and after the COVID-19 pandemic. Multiple logistic regression analysis was performed to examine which of the demographic and lifestyle factors including sitting hours per week for purposes other than study had the greatest influence on Korean adolescents' obesity status. RESULTS: The prevalence of obesity was significantly higher during the COVID-19 than before the COVID-19 (OR, 1.268, CI:1.232-1.305). There was a significant increase in the OR for sitting hours per week for purposes other than study (OR, 1.021, 95% CI, 1.019-1.024). Compared to low household income, the OR decreased for middle (OR = 0.798, 95% CI:0.77, 0.826) and high-income household students (OR, 0.833, 95% CI: 0.803-0.865). DISCUSSION/CONCLUSION: The results of this study confirmed the relationship between sit-ting hours and obesity in adolescents during the pandemic. To prevent adolescent with obesity, further studies are needed to focus on the importance of promoting health policy in adolescents to avoid the continuous rising of its prevalence and needed to understand whether the increase in obesity rates during the pandemic is a temporary trend.

Implementing Point-of-Care Ultrasound for Acute Scrotal Pain in the Pediatric Emergency Department: Screening Testicular Torsion and Patient Flow Analysis.

OBJECTIVES: Testicular torsion (TT) is a pediatric surgical emergency that requires prompt treatment. This study investigated the feasibility of point-of-care ultrasound (POCUS) for diagnosing TT in the pediatric emergency department (ED). METHODS: We retrospectively reviewed the medical records of patients, aged 18 years or younger, who visited a university-affiliated hospital pediatric ED with acute scrotal pain without trauma history and underwent diagnostic ultrasounds between January 2010 and October 2022. RESULTS: This study included 731 patients (median age: 9 years), Of these, 315 (43%) were in the POCUS-performed group: 188 in the POCUS-only group, and 127 in the POCUS-and-RADUS group. The other 416 patients (56.9%) were in the RADUS-only group. In total, 45 patients (6.2%) were diagnosed with TT (19 in the POCUS-performed group and 26 in the RADUS-only group). The sensitivity, specificity, and positive and negative predictive values of POCUS for diagnosing TT were 94.7%, 92.9%, 46.2%, and 99.6%, respectively. The median time to perform POCUS was shorter than RADUS (23 versus 61 minutes, P < .001). The POCUS-performed group had a shorter ED length of stay than the RADUS-only group (93 versus 170 minutes, P < .001). Among the patients diagnosed with TT, performing POCUS first did not significantly delay the ED process, including time to operation (250 versus 205 minutes, P = .142). CONCLUSIONS: For patients with acute scrotal pain, evaluation performed by pediatric emergency physicians using POCUS performs well in screening TT, and can decrease length of stay in the ED.

Diversity-oriented routes to thiopeptide antibiotics: total synthesis and biological evaluation of micrococcin P2.

We report the first total synthesis of micrococcin P2 (MP2, 1) by a diversity-oriented route that incorporates a number of refinements relative to earlier syntheses. Biological data regarding the activity of 1 against a range of human pathogens are also provided. Furthermore, we disclose a chemical property of MP2 that greatly facilitates medicinal chemistry work in the micrococcin area and describe a method to obtain MP2 by fermentation in B. subtilis.

Dual-Scale Doppler Attention for Human Identification.

This paper considers a Deep Convolutional Neural Network (DCNN) with an attention mechanism referred to as Dual-Scale Doppler Attention (DSDA) for human identification given a micro-Doppler (MD) signature induced as input. The MD signature includes unique gait characteristics by different sized body parts moving, as arms and legs move rapidly, while the torso moves slowly. Each person is identified based on his/her unique gait characteristic in the MD signature. DSDA provides attention at different time-frequency resolutions to cater to different MD components composed of both fast-varying and steady. Through this, DSDA can capture the unique gait characteristic of each person used for human identification. We demonstrate the validity of DSDA on a recently published benchmark dataset, IDRad. The empirical results show that the proposed DSDA outperforms previous methods, using a qualitative analysis interpretability on MD signatures.

Mitochondrial gene mutations in pediatric septic shock.

BACKGROUND: There has been a growing interest in the association between mitochondrial dysfunction and sepsis. However, most studies have focused on mitochondrial structural damage, functional aspects, or the clinical phenotypes in sepsis. The purpose of this study was to evaluate mitochondrial DNA (mtDNA) gene mutations in critically ill pediatric patients with septic shock. METHOD: Thirteen patients with severe sepsis or septic shock admitted to the pediatric intensive care unit (PICU) of a tertiary children's hospital were enrolled in this prospective observational study. Clinical data from electronic medical records were obtained. Whole-blood samples were collected within 24 h of PICU admission to perform PBMC isolation, mtDNA extraction, and mtDNA sequencing using next-generation sequencing. RESULTS: mtDNA sequencing revealed mutations in 9 of the 13 patients, presenting 27 point mutations overall, with 15 (55.6%) located in the locus related to adenosine triphosphate production and superoxide metabolism, including electron transport. CONCLUSION: In this pilot study, significant numbers of mtDNA point mutations were detected in critically ill pediatric patients with septic shock. These mutations could provide promising evidence for mitochondrial dysfunction in sepsis and a basis for further large-scale studies. IMPACT: This study is the first to examine mitochondrial DNA mutations in pediatric patients with septic shock using next-generation sequencing. A high frequency of mitochondrial DNA mutations was detected in these patients indicating an association with septic shock. This pilot study may provide a potential explanation for the association between mitochondrial dysfunction and septic shock on a genetic basis.

cAMP/EPAC Signaling Enables ETV2 to Induce Endothelial Cells with High Angiogenesis Potential.

Although the generation of ETV2-induced endothelial cells (iECs) from human fibroblasts serves as a novel therapeutic strategy in regenerative medicine, the process is inefficient, resulting in incomplete iEC angiogenesis. Therefore, we employed chromatin immunoprecipitation (ChIP) sequencing and identified molecular mechanisms underlying ETV2-mediated endothelial transdifferentiation to efficiently produce iECs retaining appropriate functionality in long-term culture. We revealed that the majority of ETV2 targets in human fibroblasts are related to vasculature development and signaling transduction pathways, including Rap1 signaling. From a screening of signaling pathway modulators, we confirmed that forskolin facilitated efficient and rapid iEC reprogramming via activation of the cyclic AMP (cAMP)/exchange proteins directly activated by cAMP (EPAC)/RAP1 axis. The iECs obtained via cAMP signaling activation showed superior angiogenesis in vivo as well as in vitro. Moreover, these cells could form aligned endothelium along the vascular lumen ex vivo when seeded into decellularized liver scaffold. Overall, our study provided evidence that the cAMP/EPAC/RAP1 axis is required for the efficient generation of iECs with angiogenesis potential.

Double-Sided Microwells with a Stepped Through-Hole Membrane for High-Throughput Microbial Assays.

To improve the throughput of microwell arrays for identifying immense cellular diversities even at a single-bacteria level, further miniaturization or densification of the microwells has been an obvious breakthrough. However, controlling millions of nanoliter samples or more at the microscale remains technologically difficult and has been spatially restricted to a single open side of the microwells. Here we employed a stepped through-hole membrane to utilize the bottom as well as top side of a high-density nanoliter microwell array, thus improving spatial efficiency. The stepped structure shows additional effectiveness for handling several millions of nanoliter bacterial samples in the overall perspectives of controllability, throughput, simplicity, versatility, and automation by using novel methods for three representative procedures in bacterial assays: partitioning cells, manipulating the chemical environment, and extracting selected cells. As a potential application, we show proof-of-concept isolation of rare cells in a mixed ratio of 1 to around 106 using a single chip. Our device can be further applied to various biological studies pertaining to synthetic biology, drug screening, mutagenesis, and single-cell heterogeneity.

Modeling of solar UV-induced photodamage on the hair follicles in human skin organoids.

Solar ultraviolet (sUV) exposure is known to cause skin damage. However, the pathological mechanisms of sUV on hair follicles have not been extensively explored. Here, we established a model of sUV-exposed skin and its appendages using human induced pluripotent stem cell-derived skin organoids with planar morphology containing hair follicles. Our model closely recapitulated several symptoms of photodamage, including skin barrier disruption, extracellular matrix degradation, and inflammatory response. Specifically, sUV induced structural damage and catagenic transition in hair follicles. As a potential therapeutic agent for hair follicles, we applied exosomes isolated from human umbilical cord blood-derived mesenchymal stem cells to sUV-exposed organoids. As a result, exosomes effectively alleviated inflammatory responses by inhibiting NF-κB activation, thereby suppressing structural damage and promoting hair follicle regeneration. Ultimately, our model provided a valuable platform to mimic skin diseases, particularly those involving hair follicles, and to evaluate the efficacy and underlying mechanisms of potential therapeutics.

CD19 CAR-expressing iPSC-derived NK cells effectively enhance migration and cytotoxicity into glioblastoma by targeting to the pericytes in tumor microenvironment.

In cancer immunotherapy, chimeric antigen receptors (CARs) targeting specific antigens have become a powerful tool for cell-based therapy. CAR-natural killer (NK) cells offer selective anticancer lysis with reduced off-tumor toxicity compared to CAR-T cells, which is beneficial in the heterogeneous milieu of solid tumors. In the tumor microenvironment (TME) of glioblastoma (GBM), pericytes not only support tumor growth but also contribute to immune evasion, underscoring their potential as therapeutic targets in GBM treatment. Given this context, our study aimed to target the GBM TME, with a special focus on pericytes expressing CD19, to evaluate the potential effectiveness of CD19 CAR-iNK cells against GBM. We performed CD19 CAR transduction in induced pluripotent stem cell-derived NK (iNK) cells. To determine whether CD19 CAR targets the TME pericytes in GBM, we developed GBM-blood vessel assembloids (GBVA) by fusing GBM spheroids with blood vessel organoids. When co-cultured with GBVA, CD19 CAR-iNK cells migrated towards the pericytes surrounding the GBM. Using a microfluidic chip, we demonstrated CD19 CAR-iNK cells' targeted action and cytotoxic effects in a perfusion-like environment. GBVA xenografts recapitulated the TME including human CD19-positive pericytes, thereby enabling the application of an in vivo model for validating the efficacy of CD19 CAR-iNK cells against GBM. Compared to GBM spheroids, the presence of pericytes significantly enhanced CD19 CAR-iNK cell migration towards GBM and reduced proliferation. These results underline the efficacy of CD19 CAR-iNK cells in targeting pericytes within the GBM TME, suggesting their potential therapeutic value for GBM treatment.

Spectral Graph Neural Network-Based Multi-Atlas Brain Network Fusion for Major Depressive Disorder Diagnosis.

Major Depressive Disorder (MDD) imposes a substantial burden within the healthcare domain, impacting millions of individuals worldwide. Functional Magnetic Resonance Imaging (fMRI) has emerged as a promising tool for the objective diagnosis of MDD, enabling the investigation of functional connectivity patterns in the brain associated with this disorder. However, most existing methods focus on a single brain atlas, which limits their ability to capture the complex, multi-scale nature of functional brain networks. To address these limitations, we propose a novel multi-atlas fusion method that incorporates early and late fusion in a unified framework. Our method introduces the concept of the holistic Functional Connectivity Network (FCN), which captures both intra-atlas relationships within individual atlases and inter-regional relationships between atlases with different brain parcellation scales. This comprehensive representation enables the identification of potential disease-related patterns associated with MDD in the early stage of our framework. Moreover, by decoding the holistic FCN from various perspectives through multiple spectral Graph Convolutional Neural Networks and fusing their results with decision-level ensembles, we further improve the performance of MDD diagnosis. Our approach is easily implemented with minimal modifications to existing model structures and demonstrates a robust performance across different baseline models. Our method, evaluated on public resting-state fMRI datasets, surpasses the current multi-atlas fusion methods, enhancing the accuracy of MDD diagnosis. The proposed novel multi-atlas fusion framework provides a more reliable MDD diagnostic technique. Experimental results show our approach outperforms both single- and multi-atlas-based methods, demonstrating its effectiveness in advancing MDD diagnosis.

Outcomes of carotid endarterectomy in octogenarians compared to their younger counterparts: a retrospective observational study.

Purpose: This study was performed to analyze the association between age and outcomes of carotid endarterectomy (CEA) by comparing postoperative outcomes between octogenarians and younger patients. Methods: From November 1994 to December 2022, 1,585 internal carotid arteries of 1,434 patients were enrolled. Patients were stratified into 2 groups: octogenarians (≥80 years old) and non-octogenarians (<80 years old). Primary endpoints were early (≤30 days) outcomes of ipsilateral stroke, any stroke, myocardial infarction, death, and major adverse cardiovascular events (MACE). We also compared overall any stroke and death between the 2 groups. Results: One of 132 octogenarians (0.8%) and 17 of 1,453 non-octogenarians (1.1%) experienced ipsilateral stroke within 30 days. Thirty-day MACE occurred in 4 of 132 octogenarians (3%) and 44 of 1,453 non-octogenarians (3%). There were no significant differences in any early (≤30 days) outcomes. Symptomatic status was associated with increased 30-day MACE (odds ratio [OR], 2.610; 95% confidence interval [CI], 1.450-4.696; P = 0.003) and 30-day any stroke (OR, 3.999; 95% CI, 1.627-9.828; P = 0.003). Symptomatic status was also associated with overall any stroke (hazard ratio [HR], 2.885; 95% CI, 1.865-4.463; P < 0.001), but age of ≥80 years was not associated with 30-day MACE, 30-day any stroke, or overall stroke. Age of ≥80 years was only associated with overall survival (HR, 2.644; 95% CI, 1.967-3.555; P < 0.001). Conclusion: CEA would be a safe and effective treatment for octogenarians with low 30-day complications and long-term stroke rates, comparable with that of younger counterparts. Advanced age is not a contraindication for CEA.

Bridging the BCI illiteracy gap: a subject-to-subject semantic style transfer for EEG-based motor imagery classification.

Introduction: Brain-computer interfaces (BCIs) facilitate direct interaction between the human brain and computers, enabling individuals to control external devices through cognitive processes. Despite its potential, the problem of BCI illiteracy remains one of the major challenges due to inter-subject EEG variability, which hinders many users from effectively utilizing BCI systems. In this study, we propose a subject-to-subject semantic style transfer network (SSSTN) at the feature-level to address the BCI illiteracy problem in electroencephalogram (EEG)-based motor imagery (MI) classification tasks. Methods: Our approach uses the continuous wavelet transform method to convert high-dimensional EEG data into images as input data. The SSSTN 1) trains a classifier for each subject, 2) transfers the distribution of class discrimination styles from the source subject (the best-performing subject for the classifier, i.e., BCI expert) to each subject of the target domain (the remaining subjects except the source subject, specifically BCI illiterates) through the proposed style loss, and applies a modified content loss to preserve the class-relevant semantic information of the target domain, and 3) finally merges the classifier predictions of both source and target subject using an ensemble technique. Results and discussion: We evaluate the proposed method on the BCI Competition IV-2a and IV-2b datasets and demonstrate improved classification performance over existing methods, especially for BCI illiterate users. The ablation experiments and t-SNE visualizations further highlight the effectiveness of the proposed method in achieving meaningful feature-level semantic style transfer.

Bioengineered liver crosslinked with nano-graphene oxide enables efficient liver regeneration via MMP suppression and immunomodulation.

Decellularized extracellular matrix scaffold, widely utilized for organ engineering, often undergoes matrix decomposition after transplantation and produces byproducts that cause inflammation, leading to clinical failure. Here we propose a strategy using nano-graphene oxide to modify the biophysical properties of decellularized liver scaffolds. Notably, we demonstrate that scaffolds crosslinked with nano-graphene oxide show high resistance to enzymatic degradation via direct inhibition of matrix metalloproteinase activity and increased mechanical rigidity. We find that M2-like macrophage polarization is promoted within the crosslinked scaffolds, which reduces graft-elicited inflammation. Moreover, we show that low activities of matrix metalloproteinases, attributed to both nano-graphene oxide and tissue inhibitors of metalloproteinases expressed by M2c, can protect the crosslinked scaffolds against in vivo degradation. Lastly, we demonstrate that bioengineered livers fabricated with the crosslinked scaffolds remain functional, thereby effectively regenerating damaged livers after transplantation into liver failure mouse models. Overall, nano-graphene oxide crosslinking prolongs allograft survival and ultimately improves therapeutic effects of bioengineered livers, which offer an alternative for donor organs.

Cortical-blood vessel assembloids exhibit Alzheimer's disease phenotypes by activating glia after SARS-CoV-2 infection.

A correlation between COVID-19 and Alzheimer's disease (AD) has been proposed recently. Although the number of case reports on neuroinflammation in COVID-19 patients has increased, studies of SARS-CoV-2 neurotrophic pathology using brain organoids have restricted recapitulation of those phenotypes due to insufficiency of immune cells and absence of vasculature. Cerebral pericytes and endothelial cells, the major components of blood-brain barrier, express viral entry receptors for SARS-CoV-2 and response to systemic inflammation including direct cell death. To overcome the limitations, we developed cortical-blood vessel assembloids by fusing cortical organoid with blood vessel organoid to provide vasculature to brain organoids a nd obtained the characteristics of increased expression of microglia and astrocytes in brain organoids. Furthermore, we observed AD pathologies, including ß-amyloid plaques, which were affected by the inflammatory response from SARS-CoV-2 infection. These findings provide an advanced platform to investigate human neurotrophic diseases, including COVID-19, and suggest that neuroinflammation caused by viral infection facilitates AD pathology.

3D microengineered vascularized tumor spheroids for drug delivery and efficacy testing.

Tumor angiogenesis is regarded as a promising target for limiting cancer progression because tumor-associated vasculature supplies blood and provides a path for metastasis. Thus, in vitro recapitulation of vascularized tumors is critical to understand the pathology of cancer and identify the mechanisms by which tumor cells proliferate, metastasize, and respond to drugs. In this study, we microengineered a vascularized tumor spheroid (VTS) model to reproduce the pathological features of solid tumors. We first generated tumor-EC hybrid spheroids with self-assembled intratumoral vessels, which enhanced the uniformity of the spheroids and peritumoral angiogenic capacity compared to spheroids composed only with cancer cells. Notably, the hybrid spheroids also exhibited expression profiles associated with aggressive behavior. The blood vessels sprouting around the hybrid spheroids on the VTS chip displayed the distinctive characteristics of leaky tumor vessels. With the VTS chip showing a progressive tumor phenotype, we validated the suppressive effects of axitinib on tumor growth and angiogenesis, which depended on exposure dose and time, highlighting the significance of tumor vascularization to predict the efficacy of anticancer drugs. Ultimately, we effectively induced both lymphangiogenesis and angiogenesis around the tumor spheroid by promoting interstitial flow. Thus, our VTS model is a valuable platform with which to investigate the interactions between tumor microenvironments and explore therapeutic strategies in cancer. STATEMENT OF SIGNIFICANCE: We conducted an integrative study within a vascularized tumor spheroid (VTS) model. We first generated tumor-EC hybrid spheroids with self-assembled intratumoral vessels, which enhanced the uniformity of the spheroids and peritumoral angiogenic capacity compared to spheroids composed only with cancer cells. Through RNA sequencing, we elucidated that the tumor-EC hybrid spheroids exhibited expression profiles associated with aggressive behavior such as cancer progression, invasion and metastasis. The blood vessels sprouting around the hybrid spheroids on the VTS chip displayed the distinctive characteristics of leaky tumor vessels. We further validated the suppressive effects of axitinib on tumor growth and angiogenesis, depending on exposure dose and time. Ultimately, we effectively induced both lymphangiogenesis and angiogenesis around the tumor spheroid by promoting interstitial flow.

Quantitative analysis of therapeutic proteins in biological fluids: recent advancement in analytical techniques.

Pharmaceutical application of therapeutic proteins has been continuously expanded for the treatment of various diseases. Efficient and reliable bioanalytical methods are essential to expedite the identification and successful clinical development of therapeutic proteins. In particular, selective quantitative assays in a high-throughput format are critical for the pharmacokinetic and pharmacodynamic evaluation of protein drugs and to meet the regulatory requirements for new drug approval. However, the inherent complexity of proteins and many interfering substances presented in biological matrices have a great impact on the specificity, sensitivity, accuracy, and robustness of analytical assays, thereby hindering the quantification of proteins. To overcome these issues, various protein assays and sample preparation methods are currently available in a medium- or high-throughput format. While there is no standard or universal approach suitable for all circumstances, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay often becomes a method of choice for the identification and quantitative analysis of therapeutic proteins in complex biological samples, owing to its high sensitivity, specificity, and throughput. Accordingly, its application as an essential analytical tool is continuously expanded in pharmaceutical R&D processes. Proper sample preparation is also important since clean samples can minimize the interference from co-existing substances and improve the specificity and sensitivity of LC-MS/MS assays. A combination of different methods can be utilized to improve bioanalytical performance and ensure more accurate quantification. This review provides an overview of various protein assays and sample preparation methods, with particular emphasis on quantitative protein analysis by LC-MS/MS.

Identification of Micrococcin P2-Derivatives as Antibiotic Candidates against Two Gram-Positive Pathogens.

Thiopeptides exhibit potent antimicrobial activity against Gram-positive pathogens by inhibiting bacterial protein synthesis. Micrococcins are among the structurally simpler thiopeptides, but they have not been exploited in detail. This research involved a computational simulation of micrococcin P2 (MP2) docking in parallel with the structure-activity relationship (SAR) studied. The incorporation of particular nitrogen heterocycles in the side chain of MP2 enhances the antimicrobial activity. Micrococcin analogues 6c and 6d thus proved to be more effective against impetigo and C. difficile infection (CDI), respectively, as compared to current first-line treatments. Compound 6c also showed a shorter treatment period than that of a first-line treatment for impetigo. This may be attributed to its ability to downregulate pro-inflammatory cytokines. Compound 6d had no observed recurrence for C. difficile and exerted a minimal impact on the beneficial gut microbiome. Their pharmacokinetic properties and low toxicity profile make these compounds ideal candidates for the treatment of impetigo and CDI and validate their involvement in preclinical development.