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The vibrational, rotational, and translational energy distributions for the CO photofragments from the 193, 248, and 308 nm photolysis of Ni(CO)4 in a supersonic molecular beam have been determined by vacuum ultraviolet laser-induced fluorescence. The measured product energy distributions appeared to be statistical, with equilibrium between the degrees of freedom investigated. The distributions were significantly colder than those calculated with a microcanonical statistical model using published bond energies. To model the measured distributions, it was necessary to postulate that the unsaturated nickel carbonyl products are formed in a stable electronically excited state. By using an excited state energy consistent with published fluorescence experiments, excellent agreement was obtained between the measured distributions and those calculated using a microcanonical statistical model. These results indicate that for 193 nm photolysis, essentially all of the Ni(CO)n products are electronically excited, with about 2.8 eV of electronic excitation. The Ni(CO)n products from 248 nm photolysis are formed in both the ground and excited states, with a 3:1 branching ratio. The data taken at 308 nm also indicate the presence of both channels, with the excited state channel still important. These results are consistent with predictions of earlier ab initio work, although the detailed mechanism is somewhat different. This earlier work is discussed in light of the present results. [ABSTRACT FROM AUTHOR]

Highly effective targeted therapies are available to treat noncommunicable chronic inflammatory skin diseases. In contrast, the exact diagnosis of noncommunicable chronic inflammatory skin diseases is complicated by its complex pathogenesis and clinical and histological overlap. Particularly, the differential diagnosis of psoriasis and eczema can be challenging in some cases, and molecular diagnostic tools need to be developed to support a gold standard diagnosis. The aim of this work was to develop a real-time PCR-based molecular classifier to distinguish psoriasis from eczema in formalin-fixed and paraffin-embedded-fixed skin samples and to evaluate the use of minimally invasive microbiopsies and tape strips for molecular diagnosis. In this study, we present a formalin-fixed and paraffin-embedded-based molecular classifier that determines the probability for psoriasis with a sensitivity/specificity of 92%/100%, respectively, and an area under the curve of 0.97, delivering comparable results to our previous published RNAprotect-based molecular classifier. The psoriasis probability, as well as levels of NOS2 expression, positively correlated with the disease hallmarks of psoriasis and negatively with eczema hallmarks. Furthermore, minimally invasive tape strips and microbiopsies were effectively used to differentiate psoriasis from eczema. In summary, the molecular classifier offers broad usage in pathology laboratories as well as outpatient settings and can support the differential diagnosis of noncommunicable chronic inflammatory skin diseases on a molecular level using formalin-fixed and paraffin-embedded tissue, microbiopsies, and tape strips., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

cfDNA is an emerging biomarker with promising uses for the monitoring of cancer or infectious disease diagnostics. This work demonstrates a new concept for an automated cfDNA extraction with nanobeads as the solid phase in a centrifugal microfluidic LabDisk. By using a combination of centrifugal and magnetic forces, we retain the nanobeads in one incubation chamber while sequentially adding, incubating and removing the sample and pre-stored buffers for extraction. As the recovery rate of the typically low concentration of cfDNA is of high importance to attain sufficient amounts for analysis, optimal beadhandling is paramount. The goal is that the cfDNA in the sample adsorbs to the solid phase completely during the binding step, is retained during washing and completely removed during elution. In this work, we improved beadhandling by optimizing the incubation chamber geometry and both frequency and temperature protocols, to maximize recovery rates. For characterization of the extraction performance, synthetic mutant DNA was spiked into human plasma samples. The LabDisk showed better reproducibility in DNA recovery rates with a standard deviation of ±13% compared to a manual approach using spin-columns (±17%) or nanobeads (±26%). The extraction of colorectal cancer samples with both the developed LabDisk and a robotic automation instrument resulted in comparable allele frequencies. Consequently, we present a highly attractive solution for an automated liquid biopsy cfDNA extraction in a small benchtop device.

Almost 2 years into the pandemic and with vaccination of children significantly lagging behind adults, long-term pediatric humoral immune responses to SARS-CoV-2 are understudied. The C19.CHILD Hamburg (COVID-19 Child Health Investigation of Latent Disease) Study is a prospective cohort study designed to identify and follow up children and their household contacts infected in the early 2020 first wave of SARS-CoV-2. We screened 6113 children < 18 years by nasopharyngeal swab-PCR in a low-incidence setting after general lockdown, from May 11 to June 30, 2020. A total of 4657 participants underwent antibody testing. Positive tests were followed up by repeated PCR and serological testing of all household contacts over 6 months. In total, the study identified 67 seropositive children (1.44%); the median time after infection at first presentation was 83 days post-symptom onset (PSO). Follow-up of household contacts showed less than 100% seroprevalence in most families, with higher seroprevalence in families with adult index cases compared to pediatric index cases (OR 1.79, P = 0.047). Most importantly, children showed sustained seroconversion up to 9 months PSO, and serum antibody concentrations persistently surpassed adult levels (ratio serum IgG spike children vs. adults 90 days PSO 1.75, P < 0.001; 180 days 1.38, P = 0.01; 270 days 1.54, P = 0.001). In a low-incidence setting, SARS-CoV-2 infection and humoral immune response present distinct patterns in children including higher antibody levels, and lower seroprevalence in families with pediatric index cases. Children show long-term SARS-CoV-2 antibody responses. These findings are relevant to novel variants with increased disease burden in children, as well as for the planning of age-appropriate vaccination strategies., (© 2022. The Author(s).)

We demonstrate detection and quantification of bacterial load with a novel microfluidic one-pot wash-free fluorescence in situ hybridization (FISH) assay in droplets. The method offers minimal manual workload by only requiring mixing of the sample with reagents and loading it into a microfluidic cartridge. By centrifugal microfluidic step emulsification, our method partitioned the sample into 210 pL (73 µm in diameter) droplets for bacterial encapsulation followed by in situ permeabilization, hybridization, and signal detection. Employing locked nucleic acid (LNA)/DNA molecular beacons (LNA/DNA MBs) and NaCl-urea based hybridization buffer, the assay was characterized with Escherichia coli , Klebsiella pneumonia , and Proteus mirabilis . The assay performed with single-cell sensitivity, a 4-log dynamic range from a lower limit of quantification (LLOQ) at ~3 × 10 3 bacteria/mL to an upper limit of quantification (ULOQ) at ~3 × 10 7 bacteria/mL, anda linearity R 2 = 0.976. The total time-to-results for detection and quantification was around 1.5 hours.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood. Minimal residual disease (MRD) monitoring is an important prognostic factor for ALL treatment response and patient stratification. MRD monitoring uses personalized real-time PCR to measure the amount of cancer cells among normal cells. Due to clonal tumor evolution or secondary rearrangement processes, MRD markers can disappear during treatment, leading to false-negative MRD results and wrong decision-making in personalized treatments. Therefore, monitoring of multiple MRD markers per patient is required. For the first time, the authors present personalized multiplex mediator probe PCR (MP PCR) for MRD monitoring in ALL. These assays can precisely quantify more MRD markers in less sample material. Therefore, clinical outcomes will be less affected by clonal tumor evolution. Personalized duplex MP PCR assays were developed for different genomic MRD markers, including immunoglobulin/T-cell receptor gene rearrangements, gene fusions, and gene deletions. One duplex assay was successfully applied in a prospective patient case and compared with hydrolysis probes. Moreover, the authors increased the multiplex level from duplex to 4-plex and still met the EuroMRD requirements for reliable quantification. In addition, the authors' MRD-MP design guidelines and multiplex workflow facilitate and accelerate MP PCR assay development. This helps the standardization of personal diagnostics., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types ( KRAS and BRAF ) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex KRAS G13D (9.82 copies/mL) and 4-plex BRAF V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R 2 ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring.

Multiplexing of analyses is essential to reduce sample and reagent consumption in applications with large target panels. In applications such as cancer diagnostics, the required degree of multiplexing often exceeds the number of available fluorescence channels in polymerase chain reaction (PCR) devices. The combination of photobleaching-sensitive and photobleaching-resistant fluorophores of the same color can boost the degree of multiplexing by a factor of 2 per channel. The only additional hardware required to create virtual fluorescence color channels is a low-cost light-emitting diode (LED) setup for selective photobleaching. Here, we present an assay concept for fluorescence color multiplexing in up to 10 channels (five standard channels plus five virtual channels) using the mediator probe PCR with universal reporter (UR) fluorogenic oligonucleotides. We evaluate the photobleaching characteristic of 21 URs, which cover the whole spectral range from blue to crimson. This comprehensive UR data set is employed to demonstrate the use of three virtual channels in addition to the three standard channels of a commercial dPCR device (blue, green, and red) targeting cancer-associated point mutations ( KRAS G12D and G12V). Moreover, a LOD (limit of detection) analysis of this assay confirms the high sensitivity of the multiplexing method ( KRAS G12D: 16 DNA copies/reaction in the standard red channel and KRAS G12V: nine DNA copies/reaction in the virtual red channel). Based on the presented data set, optimal fluorogenic reporter combinations can be easily selected for the application-specific creation of virtual channels, enabling a high degree of multiplexing at low optical and technical effort.

Spinal muscular atrophy (SMA) is a rare neurodegenerative disease leading to progressive muscular atrophy, respiratory failure, and premature death. Secondary thorax and spine deformities are frequent. In July 2017, the antisense oligonucleotide nusinersen (Spinraza) was approved for the recurrent lifelong intrathecal treatment of SMA in Europe. Lumbar punctures are challenging especially in SMA patients with severe spine deformities and after spine surgery. In the light of alternative SMA therapies that are available or are expected to be available soon and which are administered orally or via one-time infusion, an appraisal of the established therapy is significant. Discussion about which therapy is the best for each individual patient will have to include not only the safety and efficacy of data but also the application form and its burden for the patient and the health care system. Therefore, we analyzed our 3-year experiences and challenges with 478 lumbar puncture procedures in 61 pediatric SMA patients with and without spine deformities or instrumentation., Competing Interests: Dr. Johannsen reports personal fees from Avexis, personal fees from Biogen, personal fees from Roche, personal fees from Sarepta, personal fees from PTC Therapeutics, outside the submitted work. All the other authors report no conflict of interest., (Thieme. All rights reserved.)