LsMybW-encoding R2R3-MYB transcription factor is responsible for a shift from black to white in lettuce seed.

KEY MESSAGE: We identified LsMybW as the allele responsible for the shift in color from black to white seeds in wild ancestors of lettuce to modern cultivars. Successfully selected white seeds are a key agronomic trait for lettuce cultivation and breeding; however, the mechanism underlying the shift from black-in its wild ancestor-to white seeds remains uncertain. We aimed to identify the gene/s responsible for white seed trait in lettuce. White seeds accumulated less proanthocyanidins than black seeds, similar to the phenotype observed in Arabidopsis TT2 mutants. Genetic mapping of a candidate gene was performed with double-digest RAD sequencing using an F2 population derived from a cross between "ShinanoPower" (white) and "Escort" (black). The white seed trait was controlled by a single recessive locus (48.055-50.197 Mbp) in linkage group 7. Using five PCR-based markers and numerous cultivars, eight candidate genes were mapped in the locus. Only the LG7_v8_49.251Mbp_HinfI marker, employing a single-nucleotide mutation in the stop codon of Lsat_1_v5_gn_7_35020.1, was completely linked to seed color phenotype. In addition, the coding region sequences for other candidate genes were identical in the resequence analysis of "ShinanoPower" and "Escort." Therefore, we proposed Lsat_1_v5_gn_7_35020.1 as the candidate gene and designated it as LsMybW (Lactuca sativa Myb White seeds), an ortholog encoding the R2R3-MYB transcription factor in Arabidopsis. When we validated the role of LsMybW through genome editing, LsMybW knockout mutants harboring an early termination codon showed a change in seed color from black to white. Therefore, LsMybW was the allele responsible for the shift in seed color. The development of a robust marker for marker-assisted selection and identification of the gene responsible for white seeds have implications for future breeding technology and physiological analysis.

Computed Tomography Renal Volumetry Better Predicts Postoperative Donor Renal Function in Live Kidney Donor Transplantation than Renal Scintigraphy: A Comparative Study.

INTRODUCTION AND OBJECTIVES: Both computed tomography (CT) and renal scintigraphy (RS) have been used to assess vascular anatomy, renal status, and split renal function (SRF). In this study, we used a recently developed software that facilitates renal volumetric evaluations to compare RS and automated CT volumetry for assessing residual renal function and, thus, estimating postoperative renal function after donor nephrectomy. METHODS: Fifty-one cases of donor nephrectomy were analyzed. Residual renal function was estimated based on RS and CT volumetry. The correlation between the postoperative estimated glomerular filtration rate (eGFR) and expected SRF, measured using RS and three types of CT volumetry data (ellipsoid, thin-slice, and 5-mm slice data), was determined. RESULTS: The correlation coefficient between actual eGFR and expected SRF was significantly associated at each time point and modality (p < 0.0001). At any time point, the difference in correlation coefficient between RS and 5-mm volumetry was significant (p value: 0.003-0.018), whereas the differences in correlation coefficients between RS and the triaxial volume calculation, and the triaxial volume calculation and 5-mm volumetry, were generally statistically insignificant. CONCLUSIONS: Expected SRF was estimated more accurately by CT volumetric calculations (especially 5-mm slice-based volumetry) than RS.

Epistatic interactions among multiple copies of FLC genes with naturally occurring insertions correlate with flowering time variation in radish.

Brassicaceae crops, which underwent whole-genome triplication during their evolution, have multiple copies of flowering-related genes. Interactions among multiple gene copies may be involved in flowering time regulation; however, this mechanism is poorly understood. In this study, we performed comprehensive, high-throughput RNA sequencing analysis to identify candidate genes involved in the extremely late-bolting (LB) trait in radish. Then, we examined the regulatory roles and interactions of radish FLOWERING LOCUS C (RsFLC) paralogs, the main flowering repressor candidates. Seven flowering integrator genes, five vernalization genes, nine photoperiodic/circadian clock genes and eight genes from other flowering pathways were differentially expressed in the early-bolting (EB) cultivar 'Aokubinagafuto' and LB radish cultivar 'Tokinashi' under different vernalization conditions. In the LB cultivar, RsFLC1 and RsFLC2 expression levels were maintained after 40 days of cold exposure. Bolting time was significantly correlated with the expression rates of RsFLC1 and RsFLC2. Using the EB × LB F2 population, we performed association analyses of genotypes with or without 1910- and 1627-bp insertions in the first introns of RsFLC1 and RsFLC2, respectively. The insertion alleles prevented the repression of their respective FLC genes under cold conditions. Interestingly, genotypes homozygous for RsFLC2 insertion alleles maintained high RsFLC1 and RsFLC3 expression levels under cold conditions, and two-way analysis of variance revealed that RsFLC1 and RsFLC3 expression was influenced by the RsFLC2 genotype. Our results indicate that insertions in the first introns of RsFLC1 and RsFLC2 contribute to the late-flowering trait in radish via different mechanisms. The RsFLC2 insertion allele conferred a strong delay in bolting by inhibiting the repression of all three RsFLC genes, suggesting that radish flowering time is determined by epistatic interactions among multiple FLC gene copies.

Simplified approach to the medial internal iliac region using a uretero-hypogastric nerve fascia development procedure for extended pelvic lymph node dissection during robot-assisted radical prostatectomy for high-risk prostate cancer.

INTRODUCTION: Although several clinical guidelines for prostate cancer (PC) recommend extended pelvic lymph node dissection (ePLND) during radical prostatectomy for high-risk cases, there are several issues to consider, including certain technical aspects. A simplified approach to the medial internal iliac region and paravesical arteries has not been established. The uretero-hypogastric nerve fascia (UHF) envelopes the ureter, hypogastric nerve, and pelvic autonomic nerves. To preserve the UHF, it is possible to approach the medial side of the internal iliac vessels without injuring any important tissue. We analyzed technical feasibility and lymph node (LN) yields. PATIENTS AND METHODS: After obtaining institutional review board approval, 265 high-risk PC patients with ePLND were identified. A da Vinci S or Xi robotic surgical system (Intuitive Surgical, Sunnyvale, CA, USA) was used. We divided the patients into conventional (non-UHF) method and modified (UHF) groups. The numbers of LNs removed, procedure-related complications, and surgical outcomes were analyzed. RESULTS: The median number of LNs removed was 19.0 in the non-UHF group and 22.0 in the UHF group (p = 0.004). Significantly more LNs were removed from the internal iliac region in the UHF group (p = 0.042). There was no difference in overall operative, console, or LN dissection time, or the severe complication rate (Clavien-Dindo grade ≥ III), between the non-UHF and UHF groups. CONCLUSIONS: Our simplified approach using the UHF development technique is technically feasible, has no major complications, and allows for the removal of significantly more LNs compared with the conventional method.

Peritoneal invasion of prostate cancer directly confirmed during robot-assisted radical prostatectomy.

Introduction: Several studies have been published on direct rectal invasion in patients with advanced metastatic prostate cancer, but few have directly confirmed intraoperative invasion of prostate cancer into the peritoneum. Case presentation: We report the case of a 73-year-old man with prostate cancer who exhibited peritoneal invasion during robot-assisted radical prostatectomy. His prostate-specific antigen level fell to 0.38 ng/mL after surgery; he was therefore prescribed radiation and androgen-deprivation therapies that controlled the cancer for more than 1 year. Conclusions: We encountered a case showing direct peritoneal invasion of prostate cancer during robot-assisted radical prostatectomy. If invasion of the seminal vesicle is suspected, the vesicorectal fossa should be examined during robot-assisted radical prostatectomy. Preoperative confirmation by diffusion magnetic resonance imaging of the peritoneum is also useful.

[A Case of Multiple Renal Carcinomas with Three Different Subtypes].

We report a case of dialysis kidney with multiple renal carcinomas in three locations. A 74-year-old man who had a 20-year history of dialysis, was admitted to our hospital complaining of sudden right lateral area pain. Computed tomography (CT) scan revealed hemorrhage from the right dialyzed renal subcapsule. He underwent immediate transcatheter arterial embolization (TAE), but after 6 days the CT scan showed new active bleeding. A second TAE was performed, but, the CT scan several days later showed hemorrhage from the same site. So we decided to perform a right radical nephrectomy. Pathological results revealed three different renal carcinomas at sites different from the bleeding site. The presence of triple carcinomas in the same organ of the kidney is quite rare. Because of the high incidence of renal carcinoma associated with polycystic kidney disease in long-term hemodialysis patients and the possibility of multiple carcinomas as in this case, radical nephrectomy is recommended even in the case of spontaneous rupture.

Efficient Agrobacterium-mediated genetic transformation method using hypocotyl explants of radish (Raphanus sativus L.).

To investigate the gene function of radish (Raphanus sativus L.), several attempts have been made to generate genetically transformed radish. However, no efficient and relatively simple method for the genetic transformation of radish has been developed to date. In this study, we established an Agrobacterium-mediated genetic transformation method using the hypocotyl-derived explants of radish cultivar "Pirabikku". Primarily based on the Brassica transformation procedure, we optimized it for radish transformation. Using this system, the transformation efficiency of radish hypocotyl explants by Agrobacterium tumefaciens strain GV3101 harboring pIG121-Hm was 13.3%. The copy number of transfer DNA integrated into the genome was either one or two in the four independent transgenic plants. Two of the four plants exhibited male sterility and did not produce self-pollinated seeds. Examination of the expression of the ß-glucuronidase (GUS) gene in T1 plants from fertile T0 plants showed that the GUS genes were inherited. The improvement in the genetic transformation in this study might pave the way for accelerated molecular breeding and genetic analysis of radish.

Optimizing first trocar access for robot-assisted radical prostatectomy: Optical trocar access through the upper abdominal quadrant using the Kii Fios First Entry trocar.

OBJECTIVES: A pre-equipped metal trocar is required to use as a camera trocar due to a specification change in the da Vinci X/Xi system (Intuitive Surgical). We observed slight slippage of a trocar placed by the open method. With optical trocar access (OTA), the initial trocar is viewed directly with a laparoscope during placement. Reports regarding OTA for robotic surgery are limited, particularly for robot-assisted radical prostatectomy (RARP). We modified the OTA procedure such that it was appropriate for RARP. PATIENTS AND METHODS: A total of 158 patients were enrolled in this study. The first trocar placement time (FTPT) was compared between the open and OTA groups. In the OTA group, the trocar was mainly placed through the upper abdominal quadrant. We also analyzed the differences between the conventional and modified OTA procedures using the Kii Fios First Entry trocar (Applied Medical). We examined the factors affecting the FTPT using linear regression models. A P value <.05 was considered significant. RESULTS: The FTPT was significantly shorter in the OTA group than the open group (P < .0001). The modified method was associated with a shorter FTPT (P = .0001). None of the patient characteristics affected the FTPT in either group. No major complications were observed. CONCLUSIONS: OTA was applied successfully during RARP. Use of the Kii Fios First Entry trocar with upper abdominal quadrant placement was suitable for RARP.

One-surgeon basketing technique for stone extraction during flexible ureteroscopy for urolithiasis: A comparison between novice and expert surgeons.

OBJECTIVE: To determine the safety and efficacy of the one-surgeon basketing technique for stone extraction during flexible ureteroscopy when carried out by novice surgeons under instructor guidance. METHODS: We retrospectively compared perioperative results, complications and the "stone-free" rate (defined as ≤2 mm fragments on kidney-ureter-bladder imaging 1 month after flexible ureteroscopy) between experienced (group A, n = 50) and novice (group B, n = 50) surgeons using the one-surgeon basketing technique. RESULTS: Baseline patients' characteristics were similar between the two groups. There were no significant differences between groups A and B in operative time (mean 76 min vs 85 min, P = 0.46), stone-free rate (98% vs 92%, P = 0.36), postoperative fever events (4% vs 4%, P = 1.00) and postoperative hospital stay (24 h vs 24 h, P = 1.00). Clavien-Dindo grade IIIa complications (ureter injury) were only observed in two cases (4%) in group B. CONCLUSIONS: The one-surgeon basketing technique for the extraction of stone fragments during flexible ureteroscopy might be safely and effectively carried out by surgeons with no prior experience under proper guidance.

A CIN-like TCP transcription factor (LsTCP4) having retrotransposon insertion associates with a shift from Salinas type to Empire type in crisphead lettuce (Lactuca sativa L.).

To improve several agronomic traits in crisphead lettuce (Lactuca sativa L.) under high-temperature growth conditions, we investigated the correlation among those traits in multiple cultivars and performed genetic mapping of their causal genes. In a field cultivation test of Empire type (serrated leaf) and Salinas type (wavy leaf) cultivars, Empire type cultivars showed increased tipburn susceptibility and late bolting compared with Salinas type cultivars. We attempted genetic mapping of leaf shape and bolting time by ddRAD-seq using the F2 population derived from a cross between 'VI185' (Empire type) and 'ShinanoGreen' (Salinas type). These analyses suggested that both traits are controlled by a single locus in LG5. Genotyping of 51 commercial lettuce cultivars with a tightly linked marker (LG5_v8_252.743Mbp) at this locus showed an association between its genotype and the serrated leaf phenotype. By further fine mapping and transcriptome analysis, a gene encoding putative CIN-like TCP transcription factor was determined to be a candidate gene at this locus and was designated as LsTCP4. An insertion of retrotransposable element was found in the allele of 'VI185', and its transcript level in the leaves was lower than that in 'ShinanoGreen'. Because shapes of leaf epidermal cells in 'VI185' were similar to those in the TCP family mutant of Arabidopsis thaliana, the leaf shape phenotype was likely caused by reduced expression of LsTCP4. Furthermore, because it is known that the TCP family protein also controls flowering time via interaction with FT in A. thaliana, it was highly possible that LsTCP4 gave pleiotropic effects on both leaf shape and bolting time in lettuce.

Decoding ABA and osmostress signalling in plants from an evolutionary point of view.

The plant hormone abscisic acid (ABA) is fundamental for land plant adaptation to water-limited conditions. Osmostress, such as drought, induces ABA accumulation in angiosperms, triggering physiological responses such as stomata closure. The core components of angiosperm ABA signalling are soluble ABA receptors, group A protein phosphatase type 2C and SNF1-related protein kinase2 (SnRK2). ABA also has various functions in non-angiosperms, however, suggesting that its role in adaptation to land may not have been angiosperm-specific. Indeed, among land plants, the core ABA signalling components are evolutionarily conserved, implying their presence in a common ancestor. Results of ongoing functional genomics studies of ABA signalling components in bryophytes and algae have expanded our understanding of the evolutionary role of ABA signalling, with genome sequencing uncovering the ABA core module even in algae. In this review, we describe recent discoveries involving the ABA core module in non-angiosperms, tracing the footprints of how ABA evolved as a phytohormone. We also cover the latest findings on Raf-like kinases as upstream regulators of the core ABA module component SnRK2. Finally, we discuss the origin of ABA signalling from an evolutionary perspective.

Erratum: Publisher Correction: SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment.

[This corrects the article DOI: 10.1038/s42003-019-0281-1.].

SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment.

The SNF1-related protein kinase 2 (SnRK2) family includes key regulators of osmostress and abscisic acid (ABA) responses in angiosperms and can be classified into three subclasses. Subclass III SnRK2s act in the ABA response while ABA-nonresponsive subclass I SnRK2s are regulated through osmostress. Here we report that an ancient subclass III SnRK2-based signalling module including ABA and an upstream Raf-like kinase (ARK) exclusively protects the moss Physcomitrella patens from drought. Subclass III SnRK2s from both Arabidopsis and from the semiterrestrial alga Klebsormidium nitens, which contains all the components of ABA signalling except ABA receptors, complement Physcomitrella snrk2 - mutants, whereas Arabidopsis subclass I SnRK2 cannot. We propose that the earliest land plants developed the ABA/ARK/subclass III SnRK2 signalling module by recruiting ABA to regulate a pre-existing dehydration response and that subsequently a novel subclass I SnRK2 system evolved in vascular plants conferring osmostress protection independently from the ancient system.

Renal cell carcinoma treatment during pregnancy: Histopathological findings suggestive of rapid tumor growth.

INTRODUCTION: Diagnosis of renal cell carcinoma during pregnancy is rare. We report a case of renal cell carcinoma during pregnancy with rapid growth. CASE PRESENTATION: A 39-year-old woman presented to our hospital for treatment of renal tumor at 22 weeks gestation. The tumor had a cystic lesion with a partition and showed rapid growth from 28 mm to 32 mm over a period of 4 weeks. The tumor was diagnosed as renal cell carcinoma and an open partial nephrectomy was scheduled at 26 weeks gestation. The operation and perioperative course were successful. Pathological findings confirmed the tumor to be clear cell renal cell carcinoma with G2 > G3, Fuhrman grade 2, pT1a, negative surgical margin, and positive detection of progesterone receptor. CONCLUSION: We reported the successful management of a patient who was diagnosed with renal cell carcinoma during pregnancy. We also had a suggested association between rapid growth tumor and progesterone based on histopathological analysis of the tumor.

[A Case of a Congenital Right External Iliac Vein Defect Found after Robot-Assisted Radical Prostatectomy].

Congenital external iliac vein defects are extremely rare. A 56-year-old man underwent robotic prostatectomy (RARP) without pelvic lymph node dissection (PLND) for localized low-risk prostate cancer. Intraoperative findings revealed a markedly dilated vein on the dorsal side of the pubic bone,which was difficult to preserve and was therefore clipped. Sudden swelling and pain appeared in the right leg on day 2 after successful RARP. A contrast-enhanced computed tomography (CT) scan showed a deep vein thrombus (DVT) and lack of a right external iliac vein. Immediate heparinization improved the symptoms, and no other postoperative complications occurred. In the retrospective review,a large dilated vein lying transversely on the prostate surface was observed with a robotic scope,but no manipulation of the pelvic vessels was performed because PLND was not performed. Therefore,the dilated vein was not recognized as a shunt from the right femoral vein to the left external iliac vein. A preoperative staging CT scan,which faintly revealed the right external iliac vein,could be useful to identify this extremely rare deformity in advance of surgery. Although preoperative imaging examinations are often difficult to diagnose preoperatively,as in this case,we consider it important to use appropriate approaches and treatment when dealing with symptoms that arise during pelvic surgery in patients with many anatomical variations.

Archetypal Roles of an Abscisic Acid Receptor in Drought and Sugar Responses in Liverworts.

Abscisic acid (ABA) controls seed dormancy and stomatal closure through binding to the intracellular receptor Pyrabactin resistance1 (Pyr1)/Pyr1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) in angiosperms. Genes encoding PYR/PYL/RCAR are thought to have arisen in the ancestor of embryophytes, but the roles of the genes in nonvascular plants have not been determined. In the liverwort Marchantia polymorpha, ABA reduces growth and enhances desiccation tolerance through increasing accumulation of intracellular sugars and various transcripts such as those of Late Embryogenesis Abundant (LEA)-like genes. In this study, we analyzed a gene designated MpPYL1, which is closely related to PYR/PYL/RCAR of angiosperms, in transgenic liverworts. Transgenic lines overexpressing MpPYL1-GFP showed ABA-hypersensitive growth with enhanced desiccation tolerance, whereas Mppyl1 generated by CRISPR-Cas9-mediated genome editing showed ABA-insensitive growth with reduced desiccation tolerance. Transcriptome analysis indicated that MpPYL1 is a major regulator of abiotic stress-associated genes, including all 35 ABA-induced LEA-like genes. Furthermore, these transgenic plants showed altered responses to extracellular Suc, suggesting that ABA and PYR/PYL/RCAR function in sugar responses. The results presented here reveal an important role of PYR/PYL/RCAR in the ABA response, which was likely acquired in the common ancestor of land plants. The results also indicate the archetypal role of ABA and its receptor in sugar response and accumulation processes for vegetative desiccation tolerance in bryophytes.

Enhancing analysis throughput, sensitivity and specificity in LC/ESI-MS/MS assay of plasma 25-hydroxyvitamin D3 by derivatization with triplex 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) isotopologues.

The plasma/serum concentration of 25-hydroxyvitamin D3 [25(OH)D3] is a diagnostic index for vitamin D deficiency/insufficiency, which is associated with a wide range of diseases, such as rickets, cancer and diabetes. We have reported that the derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) works well in the liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) assay of the serum/plasma 25(OH)D3 for enhancing the sensitivity and the separation from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3]. However, enhancing the analysis throughput remains an issue in the LC/ESI-MS/MS assay of 25(OH)D3. The most obvious restriction of the LC/MS/MS throughput is the chromatographic run time. In this study, we developed an enhanced throughput method for the determination of the plasma 25(OH)D3 by LC/ESI-MS/MS combined with the derivatization using the triplex (2H0-, 2H3- and 2H6-) DAPTAD isotopologues. After separate derivatization with 1 of 3 different isotopologues, the 3 samples were combined and injected together into LC/ESI-MS/MS. Based on the mass differences between the isotopologues, the derivatized 25(OH)D3 in the 3 different samples were quantified within a single run. The developed method tripled the hourly analysis throughput without sacrificing assay performance, i.e., ease of pretreatment of plasma sample (only deproteinization), limit of quantification (1.0ng/mL when a 5µL-plasma was used), precision (intra-assay RSD≤5.9% and inter-assay RSD≤5.5%), accuracy (98.7-102.2%), matrix effects, and capability of separating from an interfering metabolite, 3-epi-25(OH)D3. The multiplexing of samples by the isotopologue derivatization was applied to the analysis of plasma samples of healthy subjects and the developed method was proven to have a satisfactory applicability.

Stimulation of cellular senescent processes, including secretory phenotypes and anti-oxidant responses, after androgen deprivation therapy in human prostate cancer.

Endocrine resistance is a major problem in prostate cancer. Recent studies suggest that cellular plasticity plays a key role in therapy resistance. Yet little is known about the cellular changes of human prostate cancer after androgen deprivation therapy (ADT). In this study, we investigated cellular senescence, senescence-associated secretory phenotypes (SASPs), and anti-oxidant responses. Hormone ablation upregulated senescence-associated (SA)-ß-Gal activity in prostate glands, as well as the expressions of p27KIP1 and p53, in a mouse castration model. In line with this, the expressions of p21CIP1 and p27KIP1 were significantly more upregulated in human non-pathological prostatic glands after ADT than in untreated specimens. In a study of SASP markers, the expressions of IL6 and IL8 were also more upregulated in human non-pathological prostatic glands after ADT than in untreated specimens. IL6, IL8, and MMP2 were expressed more strongly in human prostate cancer specimens resected after ADT than in untreated tumors. Of note, treatment with the anti-oxidant reagent NAC significantly suppressed SA-ß-Gal activity in androgen-sensitive human prostate cancer LNCaP cells. In immunohistochemical analyses on anti-oxidant response genes, NRF2 and NQO1 were more upregulated after hormone ablation in human prostate gland and carcinoma specimens after ADT than in untreated specimens or in murine prostate glands after castration. Taken together, these findings suggest that ADT induces cellular senescence processes accompanied by secretory phenotypes and anti-oxidant responses in prostate. These cellular changes may be attractive targets for preventing endocrine resistance in prostate cancer.

Oxygen delivery using neonatal self-inflating bags without reservoirs.

BACKGROUND: Guidelines recommend avoiding excessive oxygen during neonatal resuscitation. Recent studies have suggested that oxygen titration can be achieved using a self-inflating bag, but data on the effectiveness of resuscitators used in neonatal ventilation are scarce, The aim of this study was therefore to determine the amount of oxygen delivered using several brands of neonatal self-inflating resuscitation bags without reservoirs under different conditions with regard to oxygen flow rate, ventilation rate (VR), peak inspiratory pressure (PIP) range, and test lung compliance. METHODS: Oxygen concentration was measured under a variety of conditions. Combinations of oxygen flow rate (10, 5.0, 3.0 and 1.0 L/min), VR (40, 60 inflations/min), PIP range (20-25 cmH2 O, 35-40 cmH2 O), and test lung compliance (0.6, 1.0, 3.0, and 5.0 mL/cmH2 O) were examined using six kinds of self-inflating bag. RESULTS: Delivered oxygen concentration varied widely (30.1-96.7%) and had a significant positive correlation with gas flow rate in all of the bags. Delivered oxygen concentration was also negatively correlated with PIP in all of the bags and with VR in some of them. Test lung compliance did not affect delivered oxygen concentration. CONCLUSION: The use of neonatal resuscitation self-inflating bags without reservoirs resulted in different delivered oxygen concentrations depending on gas flow rate, VR, PIP, and manufacturer, but not on lung compliance. This suggests that targeted oxygen concentrations could be delivered, even in lungs with decreased compliance, during resuscitation.

A Method for Simultaneous Determination of 25-Hydroxyvitamin D3 and Its 3-Sulfate in Newborn Plasma by LC/ESI-MS/MS after Derivatization with a Proton-Affinitive Cookson-Type Reagent.

A method for the simultaneous determination of 25-hydroxyvitamin D3 [25(OH)D3] and its 3-sulfate [25(OH)D3S] in newborn plasma, which is expected to be helpful in the assessment of the vitamin D status, using stable isotope-dilution liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was pretreated based on the deproteinization and solid-phase extraction, then subjected to derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). The derivatization enabled the accurate quantification of 25(OH)D3 without interference from 3-epi-25(OH)D3 and also facilitated the simultaneous determination of the two metabolites by LC/positive ESI-MS/MS. Quantification was based on the selected reaction monitoring with the characteristic fragmentation of the DAPTAD-derivatives during MS/MS. This method was reproducible (intra- and inter-assay relative standard deviations of 7.8% or lower for both metabolites) and accurate (analytical recovery, 95.4-105.6%). The limits of quantification were 1.0 ng/mL and 2.5 ng/mL for 25(OH)D3 and 25(OH)D3S, respectively, when using a 20-µL sample. The developed method was applied to the simultaneous determination of plasma 25(OH)D3 and 25(OH)D3S in newborns; it was recognized that the plasma concentration of 25(OH)D3S is significantly higher than that of 25(OH)D3, and preterm newborns have lower plasma 25(OH)D3S concentrations than full-term newborns.