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• First established reference intervals for estradiol using a UHPLC-MS/MS-based method in South Korea. • Serum samples from 634 pediatric patients, including neonates, infants, children, and adolescents. • The highly analytical sensitive UHPLC-MS/MS method was useful for estradiol determination in children. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) is a reliable and accurate method for measuring steroid hormone levels. There is an increasing need for sensitive and precise methods to measure estradiol in pediatric patients. Here, we established reference intervals for estradiol in healthy children using a UHPLC–MS/MS-based method for the first time in South Korea. Serum estradiol was measured using a Sciex Triple QuadTM 6500 + UHPLC–MS/MS (Sciex, Framingham, MA, USA). Reference intervals for estradiol were established according to the CLSI document EP28-A3c:2008. The reference intervals were validated using serum samples from 634 pediatric patients, including neonates, children, and adolescents. Among them, 389 specimens were used in analysis of the specimen acceptance time. Statistical analysis was performed using MedCalc (MedCalc, Ostend, Belgium) and Analyse–it (Analyse–it Software Ltd., Leeds, United Kingdom) software. Reference intervals for boys (n = 297) were <16.6, <7.3, <19.0, <30.5, 7.6–96.5, and 10.6–134.4 pmol/L among those aged <1, 1–5, 6–9, 10–11, 12–14, and 15–17 years, respectively. Reference intervals for girls (n = 337) were <114.7, <24.2, <34.8, 8.0–177.0, 10.4–480.5, and 9.1–486.7 pmol/L among those aged <1, 1–5, 6–9, 10–11, 12–14, and 15–17 years, respectively. Overall, there was no effect of specimen acceptance time on estradiol measurements in boys or girls, except for that in the group aged 10–11 years. The reference intervals for healthy children were validated using a UHPLC–MS/MS-based method. The highly analytical sensitive UHPLC–MS/MS method may be useful for estradiol determination in pediatric patients. [ABSTRACT FROM AUTHOR]

There is an increased need for the sensitive and accurate measurement of estradiol levels in patients with estradiol-related endocrine disorders. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a reliable and accurate method for measuring steroid hormone levels. Here, we aimed to establish an LC-MS/MS-based method to quantify estradiol levels without sample derivatization and evaluated its analytical performance. Sciex Triple Quad 6500 + LC-MS/MS was used for estradiol analysis. We evaluated its analytical performance, including linearity, precision, the lower limit of detection and quantification (LoD and LoQ, respectively), accuracy, and carryover. The estradiol results determined by LC-MS/MS were compared with those obtained using a chemiluminescent microparticle immunoassay. The LC-MS/MS output was linear for serum estradiol concentrations in the range of 0.2–10311.6 pmol/L. The intra-laboratory precision (coefficient of variation) was 3.0–10.1 %. The LoD and LoQ were 2.8 and 7.5 pmol/L, respectively. The overall accuracy was within 15 % of bias, and the carryover was within the acceptable range (<1.0 %). The results of the estradiol analysis determined by LC-MS/MS were comparable to those obtained by the chemiluminescent microparticle immunoassay (r2 = 0.9843), although there was a negative bias of − 17.82 (95 % confidence interval, −27.21 to − 8.44). A highly sensitive, derivatization-free LC-MS/MS method was successfully developed in this study. This may be beneficial for estradiol measurements in patients with estradiol-related endocrine disorders. [ABSTRACT FROM AUTHOR]

Amikacin is a first-line drug that must be evaluated when performing an antimycobacterial susceptibility test (AST) for Mycobacterium avium complex (MAC). However, the presence of sporadic trailing growth in MAC makes determining the precise point for reading its minimal inhibitory concentration (MIC) challenging. Susceptibility was re-tested for 134 MAC clinical isolates using the Sensititre SLOMYCOI panel, the rrs gene was sequenced, and amikacin exposure history was investigated. The MIC 50 , MIC 90 , and the epidemiological cut-off value (ECOFF) were calculated using the EUCAST method. After re-testing and ignoring trailing growth, of the 22 M. intracellulare isolates originally classified as resistant to amikacin according to the CLSI guideline, 10 strains were reclassified as intermediate and four as susceptible. Similarly, from the seven resistant M. avium strains, one was reclassified as intermediate and four as susceptible. No rrs gene mutations were detected in any isolates, including resistant strains. When ignoring trailing growth, the calculated MIC 50 , MIC 90 , and ECOFF values closely aligned with the EUCAST MIC distribution. To maintain the current CLSI breakpoint, trailing growth should be ignored when reading the amikacin MIC of MAC. To read the MIC at complete bacterial inhibition, the CLSI breakpoint needs to be raised. [ABSTRACT FROM AUTHOR]

Objective: We aimed to determine whether combinations of multiplex cytokine responses could differentiate Mycobacterium tuberculosis (Mtb) infection states.Methods: Mtb-specific antigen-induced and unstimulated cytokines were measured by Luminex assay in supernatants of QuantiFERON® Gold In-Tube assay (QFT) in 48 active pulmonary TB patients (TB), 15 latent TB infection subjects (LTBI), and 13 healthy controls (HCs).Results: Among the 29 cytokines, eight Mtb antigen-specific biomarkers (GM-CSF, IFN-γ, IL-1RA, IL-2, IL-3, IL-13, IP-10, and MIP-1β) in the Mtb-infected group were significantly different from those of the HCs. Five Mtb-specific biomarkers (EGF, GM-CSF, IL-5, IL-10, and VEGF), two unstimulated biomarkers (TNF-α[Nil] and VEGF[Nil]), and one Mtb-specific biomarker ratio (IL-2/IFN-γ) showed significant differences between active TB and LTBI. Three unstimulated biomarkers (IL-8[Nil], IL-13[Nil], and VEGF[Nil]) and 5 Mtb-specific biomarkers (IFN-γ, IL-2, IL-3, IP-10, and VEGF) were significantly different between active TB and non-active TB groups. Combinations of three cytokine biomarkers resulted in the accurate prediction of 92.1-93.7% of Mtb-infected cases and 92.3-100% of HCs, respectively. Moreover, combinations of five biomarkers accurately predicted 90.9-100% of active TB cases and 80-100% of LTBI subjects, respectively. In discriminating between active TB and non-active TB regardless of QFT results, combinations of six biomarkers predicted 79.2-95.8% of active TB cases and 67.9-89.3% of non-active TB subjects.Conclusions: Taken together, our data suggest that combinations of whole blood Mtb antigen-dependent cytokines could serve as biomarkers to determine TB disease states. Especially, VEGF is highlighted as a key biomarker for reflecting active TB, irrespective of stimulation. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondrial DNA has been used to investigate phylogenetic relationships and pathophysiologic roles in aging, degenerative diseases, and cancer. We investigated the prognostic usefulness of mitochondrial DNA minisatellite (mtMS) markers compared with nuclear short tandem repeat markers by evaluating the laboratory performance and clinical value of these markers in a large sample of patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) with various simulated conditions in vitro and in serial follow-up samples. We examined the value of mtMS markers as a prognostic indicator in 100 patients with various hematologic disorders undergoing allo-HSCT, including 35 patients with longitudinal follow-up for 55 months. The mtMS markers showed high sensitivity and accuracy for the quantitative detection of chimerism compared with nuclear short tandem repeat markers, particularly in unrelated transplantation and under inappropriate sampling conditions. Longitudinal follow-up after allo-HSCT disclosed that chimerism precisely reflected the status of engraftment or relapse during the clinicopathological course. Moreover, changes in mtMS markers in recipients before allo-HSCT were associated with clinical outcomes. Our data indicate that mtMS markers have multiple functions in monitoring mixed chimerism and predicting prognosis after allo-HSCT. [Copyright &y& Elsevier]

It is challenging to diagnose patients with pancreatic ductal adenocarcinoma (PDAC) early on, and their treatment is often complex. Gemcitabine (GEM) is the first-line treatment for PDAC, but its efficacy is limited in most patients due to the GEM resistance from KRAS and P53 gene mutations. We describe the correction of a double gene mutation and therapeutic effect for the GEM resistant PDAC. Bio-available nanoliposomes (NL) possessing Cas9-ribonucleoproteins and adenine-base editors were developed to conduct KRAS and P53 mutation gene editing directly. NLs were conjugated with EGFR antibodies to tumor-specific delivery, and the anti-cancer effect was verified in vitro and in vivo Model. Our GEM-combinatorial therapeutic strategies using double gene editing systems with one-shot may be a potent therapy for PDAC, overcoming chemoresistance. [ABSTRACT FROM AUTHOR]

Compared to a plasmid, viral, and other delivery systems, direct Cas9/sgRNA protein delivery has several advantages such as low off-targeting effects and non-integration, but it still has limitations due to low transfer efficiency. As such, the CRISPR/Cas9 system is being developed in combination with nano-carrier technology to enhance delivery efficiency and biocompatibility. We designed a microbubble-nanoliposomal particle as a Cas9/sgRNA riboprotein complex carrier, which effectively facilitates local delivery to a specific site when agitated by ultrasound activation. In practice, we successfully transferred the protein constructs into dermal papilla cells in the hair follicle of androgenic alopecia animals by microbubble cavitation induced sonoporation of our particle. The delivered Cas9/sgRNA recognized and edited specifically the target gene with high efficiency in vitro and in vivo , thus recovering hair growth. We demonstrated the topical application of ultrasound-activated nanoparticles for androgenic alopecia therapy through the suppression of SRD5A2 protein production by CRISPR-based genomic editing. Image 1 [ABSTRACT FROM AUTHOR]

This study compared the accuracy of a new MALDI-TOF mass spectrometry system, ASTA MicroIDSys system, with that of MALDI Biotyper system for the identification of reference and clinical bacterial and yeast strains. The identification accuracy of the 2 systems was compared using a total of 406 strains comprising 142 aerobic and 180 anaerobic bacterial strains and 84 yeast strains. The genus and species identification rates were 98.0% and 89.4% using MicroIDSys and 96.1% and 89.4% using Biotyper, respectively. The species identification rates of MicroIDSys and Biotyper for aerobic bacteria were 93.0% and 97.2%, respectively, and those for anaerobic bacteria were 85.6% and 81.7%, respectively. The accuracy of yeast identification at the species level was 91.7% using MicroIDSys and 92.9% using Biotyper. These findings indicate that MicroIDSys could be useful for the accurate identification of bacteria and yeast in clinical microbiology laboratories. [ABSTRACT FROM AUTHOR]