RAD51-WSS1-dependent genetic pathways are essential for DNA-protein crosslink repair and pathogenesis in Candida albicans.

Genetic analyses in Saccharomyces cerevisiae suggest that nucleotide excision repair (NER), homologous recombination (HR), and protease-dependent repair pathways coordinately function to remove DNA-protein crosslinks (DPCs) from the genome. DPCs are genomic cytotoxic lesions generated because of the covalent linkage of proteins with DNA. Although NER and HR processes have been studied in pathogenic Candida albicans, their roles in DPC repair (DPCR) are yet to be explored. Proteases like Wss1 and Tdp1 (tyrosyl-DNA phosphodiesterase-1) are known to be involved in DPCR; however, Tdp1 that selectively removes topoisomerase-DNA complexes is intrinsically absent in C. albicans. Therefore, the mechanism of DPCR might have evolved differently in C. albicans. Herein, we investigated the interplay of three genetic pathways and found that RAD51-WSS1-dependent HR and protease-dependent repair pathways are essential for DPC removal, and their absence caused an increased rate of loss of heterozygosity in C. albicans. RAD1 but not RAD2 of NER is critical for DPCR. In addition, we observed truncation of chromosome #6 in the cells defective in both RAD51 and WSS1 genes. While the protease and DNA-binding activities are essential, a direct interaction of Wss1 with the eukaryotic DNA clamp proliferating cell nuclear antigen is not a requisite for the function of Wss1. DPCR-defective C. albicans cells exhibited filamentous morphology, reduced immune cell evasion, and attenuation in virulence. Thus, we concluded that RAD51-WSS1-dependent DPCR pathways are essential for genome stability and candidiasis development. Since no vaccine against candidiasis is available for human use yet, we propose to explore DPCR-defective attenuated strains (rad51ΔΔwss1ΔΔ and rad2ΔΔrad51ΔΔwss1ΔΔ) for whole-cell vaccine development.

Universal digital high-resolution melting for the detection of pulmonary mold infections.

Invasive mold infections (IMIs) are associated with high morbidity, particularly in immunocompromised patients, with mortality rates between 40% and 80%. Early initiation of appropriate antifungal therapy can substantially improve outcomes, yet early diagnosis remains difficult to establish and often requires multidisciplinary teams evaluating clinical and radiological findings plus supportive mycological findings. Universal digital high-resolution melting (U-dHRM) analysis may enable rapid and robust diagnoses of IMI. A universal fungal assay was developed for U-dHRM and used to generate a database of melt curve signatures for 19 clinically relevant fungal pathogens. A machine learning algorithm (ML) was trained to automatically classify these pathogen curves and detect novel melt curves. Performance was assessed on 73 clinical bronchoalveolar lavage samples from patients suspected of IMI. Novel curves were identified by micropipetting U-dHRM reactions and Sanger sequencing amplicons. U-dHRM achieved 97% overall fungal organism identification accuracy and a turnaround time of ~4 hrs. U-dHRM detected pathogenic molds (Aspergillus, Mucorales, Lomentospora, and Fusarium) in 73% of 30 samples classified as IMI, including mixed infections. Specificity was optimized by requiring the number of pathogenic mold curves detected in a sample to be >8 and a sample volume to be 1 mL, which resulted in 100% specificity in 21 at-risk patients without IMI. U-dHRM showed promise as a separate or combination diagnostic approach to standard mycological tests. U-dHRM's speed, ability to simultaneously identify and quantify clinically relevant mold pathogens in polymicrobial samples, and detect emerging opportunistic pathogens may aid treatment decisions, improving patient outcomes. IMPORTANCE: Improvements in diagnostics for invasive mold infections are urgently needed. This work presents a new molecular detection approach that addresses technical and workflow challenges to provide fast pathogen detection, identification, and quantification that could inform treatment to improve patient outcomes.

Fitness trade-offs of multidrug efflux pumps in Escherichia coli K-12 in acid or base, and with aromatic phytochemicals.

Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness trade-offs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with carbonyl cyanide 3-chlorophenylhydrazone or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemical's lipophilicity (logP). Methyl salicylate and salicylamide selected strongly against AcrA, without genetic induction of drug resistance regulons. MdtEF-TolC and EmrAB-TolC each had a fitness cost at pH 5.5, but salicylate or benzoate made the fitness contribution positive. Pump fitness effects were not explained by gene expression (measured by digital PCR). Between pH 5.5 and 8.0, acrA and emrA were upregulated in the log phase, whereas mdtE expression was upregulated in the transition-to-stationary phase and at pH 5.5 in the log phase. Methyl salicylate did not affect pump gene expression. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without inducing antibiotic resistance regulons.IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate drug resistance in our gut microbiome. Bacteria maintain low-level resistance by proton-motive force (PMF)-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring resistance to antimicrobial agents at first exposure while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference (ΔpH), which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. Non-acidic aromatic molecules have the advantage that they avoid the Mar-dependent induction of regulons conferring other forms of drug resistance. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers.

SARS-CoV-2 in digital era: Diagnostic techniques and importance of nucleic acid quantification with digital PCRs.

Studies related to clinical diagnosis and research of SARS-CoV-2 are important in the current pandemic era. Although molecular biology has emphasised the importance of qualitative analysis, quantitative analysis with nucleic acids in relation to SARS-CoV-2 needs to be clearly emphasised, which can provide perspective for viral dynamic studies of SARS-CoV-2. In this regard, the requirement and utilization of digital PCR in COVID-19 research has substantially increased during the pandemic, necessitating the aggregation of its cardinal applications and future scopes. Hence, this meta-review comprehensively addresses and emphasises the importance of nucleic acid quantification of SARS-CoV-2 RNA with digital PCR (dPCR). Various quantitative techniques of clinical significance like immunological, proteomic and nucleic acid-based diagnosis and quantification, have been comparatively discussed. Furthermore, the core part of the article focusses on the working principle and advantages of digital PCR, along with its applications in COVID-19 research. Several important applications like viral load quantitation, environmental surveillance and assay validation have been extensively investigated and discussed. Certain key future scopes of clinical importance, like mortality prediction, viral/variant-symbiosis, and antiviral studies were also identified, suggesting several possible digital PCR applications in COVID-19 research.

Development of a nine-variant reference material panel to standardize cell-free DNA detection.

Detection of specific gene mutations in cell-free DNA (cfDNA) serves as a valuable cancer biomarker and is increasingly being explored as an appealing alternative to tissue-based methods. However, the lack of available reference materials poses challenges in accurately evaluating the performance of different assays. In this study, we present the development of a comprehensive reference material panel for cfDNA detection, encompassing nine hotspot mutations in KRAS/BRAF/EGFR/PIK3CA at three variant allele frequencies (VAFs), ranging from 0.33 to 23.9%. To mimic cfDNA, these reference materials were generated by enzymatically digesting cell-line DNA into approximately 154-bp to 173-bp fragments using a laboratory-developed reaction system. The VAFs for each variation were precisely determined through validated digital PCR assays with high accuracy. Furthermore, the reliability and applicability of this panel were confirmed through two independent NGS assays, yielding concordant results. Collectively, our findings suggest that this novel reference material panel holds great potential for validation, evaluation, and quality control processes associated with liquid biopsy assays.

Assessment of Key Factors Impacting Variability in AAV Vector Genome Titration by Digital PCR.

Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze-thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.

Digital PCR Applications in the SARS-CoV-2/COVID-19 Era: a Roadmap for Future Outbreaks.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global public health disaster. The current gold standard for the diagnosis of infected patients is real-time reverse transcription-quantitative PCR (RT-qPCR). As effective as this method may be, it is subject to false-negative and -positive results, affecting its precision, especially for the detection of low viral loads in samples. In contrast, digital PCR (dPCR), the third generation of PCR, has been shown to be more effective than the gold standard, RT-qPCR, in detecting low viral loads in samples. In this review article, we selected publications to show the broad-spectrum applications of dPCR, including the development of assays and reference standards, environmental monitoring, mutation detection, and clinical diagnosis of SARS-CoV-2, while comparing it analytically to the gold standard, RT-qPCR. In summary, it is evident that the specificity, sensitivity, reproducibility, and detection limits of RT-dPCR are generally unaffected by common factors that may affect RT-qPCR. As this is the first time that dPCR is being tested in an outbreak of such a magnitude, knowledge of its applications will help chart a course for future diagnosis and monitoring of infectious disease outbreaks.

Vibrio-Sequins - dPCR-traceable DNA standards for quantitative genomics of Vibrio spp.

BACKGROUND: Vibrio spp. are a diverse group of ecologically important marine bacteria responsible for several foodborne outbreaks of gastroenteritis around the world. Their detection and characterization are moving away from conventional culture-based methods towards next generation sequencing (NGS)-based approaches. However, genomic methods are relative in nature and suffer from technical biases arising from library preparation and sequencing. Here, we introduce a quantitative NGS-based method that enables the quantitation of Vibrio spp. at the limit of quantification (LOQ) through artificial DNA standards and their absolute quantification via digital PCR (dPCR). RESULTS: We developed six DNA standards, called Vibrio-Sequins, together with optimized TaqMan assays for their quantification in individually sequenced DNA libraries via dPCR. To enable Vibrio-Sequin quantification, we validated three duplex dPCR methods to quantify the six targets. LOQs were ranging from 20 to 120 cp/µl for the six standards, whereas the limit of detection (LOD) was ~ 10 cp/µl for all six assays. Subsequently, a quantitative genomics approach was applied to quantify Vibrio-DNA in a pooled DNA mixture derived from several Vibrio species in a proof-of-concept study, demonstrating the increased power of our quantitative genomic pipeline through the coupling of NGS and dPCR. CONCLUSIONS: We significantly advance existing quantitative (meta)genomic methods by ensuring metrological traceability of NGS-based DNA quantification. Our method represents a useful tool for future metagenomic studies aiming at quantifying microbial DNA in an absolute manner. The inclusion of dPCR into sequencing-based methods supports the development of statistical approaches for the estimation of measurement uncertainties (MU) for NGS, which is still in its infancy.

The Molecular Tumor Board of the Regina Elena National Cancer Institute: from accrual to treatment in real-world.

BACKGROUND: Molecular Tumor Boards (MTB) operating in real-world have generated limited consensus on good practices for accrual, actionable alteration mapping, and outcome metrics. These topics are addressed herein in 124 MTB patients, all real-world accrued at progression, and lacking approved therapy options. METHODS: Actionable genomic alterations identified by tumor DNA (tDNA) and circulating tumor DNA (ctDNA) profiling were mapped by customized OncoKB criteria to reflect diagnostic/therapeutic indications as approved in Europe. Alterations were considered non-SoC when mapped at either OncoKB level 3, regardless of tDNA/ctDNA origin, or at OncoKB levels 1/2, provided they were undetectable in matched tDNA, and had not been exploited in previous therapy lines. RESULTS: Altogether, actionable alterations were detected in 54/124 (43.5%) MTB patients, but only in 39 cases (31%) were these alterations (25 from tDNA, 14 from ctDNA) actionable/unexploited, e.g. they had not resulted in the assignment of pre-MTB treatments. Interestingly, actionable and actionable/unexploited alterations both decreased (37.5% and 22.7% respectively) in a subset of 88 MTB patients profiled by tDNA-only, but increased considerably (77.7% and 66.7%) in 18 distinct patients undergoing combined tDNA/ctDNA testing, approaching the potential treatment opportunities (76.9%) in 147 treatment-naïve patients undergoing routine tDNA profiling for the first time. Non-SoC therapy was MTB-recommended to all 39 patients with actionable/unexploited alterations, but only 22 (56%) accessed the applicable drug, mainly due to clinical deterioration, lengthy drug-gathering procedures, and geographical distance from recruiting clinical trials. Partial response and stable disease were recorded in 8 and 7 of 19 evaluable patients, respectively. The time to progression (TTP) ratio (MTB-recommended treatment vs last pre-MTB treatment) exceeded the conventional Von Hoff 1.3 cut-off in 9/19 cases, high absolute TTP and Von Hoff values coinciding in 3 cases. Retrospectively, 8 patients receiving post-MTB treatment(s) as per physician's choice were noted to have a much longer overall survival from MTB accrual than 11 patients who had received no further treatment (35.09 vs 6.67 months, p = 0.006). CONCLUSIONS: MTB-recommended/non-SoC treatments are effective, including those assigned by ctDNA-only alterations. However, real-world MTBs may inadvertently recruit patients electively susceptible to diverse and/or multiple treatments.

Absolute quantification of SARS-CoV-2 with Clarity Plus™ digital PCR.

In recent years, the usage of digital polymerase chain reaction (dPCR) for various clinical applications has increased exponentially. In this study, a dPCR assay optimized on the Clarity Plus™ dPCR system was evaluated for the absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the global coronavirus disease 2019 (COVID-19) outbreak. The assay demonstrated good inter- and intra- assay precision, accuracy, as well as excellent linearity across a range of over 6 orders of magnitude for target gene quantification. In addition, a comparison of the assay on both dPCR and qPCR platforms revealed that dPCR exhibited a slightly higher sensitivity compared to its qPCR counterpart when quantifying SARS-CoV-2 at a lower concentration. Overall, the results showed that the dPCR assay is a reliable and effective approach for the absolute quantification of SARS-CoV-2 and can be a valuable molecular tool in clinical applications such as detecting low viral loads in patients as well as in wastewater surveillance of COVID-19.

Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA.

The development of an HIV-1 cure is hampered by the existence of a persistent (latent) reservoir that contains a small proportion of replication-competent intact proviruses which refuels viral replication upon treatment discontinuation. Therefore, an accurate evaluation and quantification of these (intact) proviruses is essential to determine the efficacy of HIV-1 cure strategies which aim to eliminate this reservoir. Here, we present two triplex digital PCR assays which resulted from a combination of two existing methods, the IPDA (a 2-colour digital PCR based method) and Q4PCR assays (4 colour qPCR method), and tested the functionality on a three-colour digital PCR platform. In the present paper, we provide a step-by-step experimental protocol for these triplex digital PCR assays and validate their performance on a latently infected Jurkat cell-line model and HIV-1 patient samples. Our data demonstrates the potential and flexibility of increasing the number of subgenomic regions of HIV-1 within the IPDA to acquire sensitive detection of the HIV-1 reservoir while benefitting from the advantages of a dPCR setup.

Establishment and evaluation of digital PCR methods for HER2 copy number variation in breast cancer.

Accurate measurement of human epidermal growth factor receptor 2 (HER2) copy number variation (CNV) is very important for guiding the tumor target therapy in breast cancer. Digital PCR (dPCR) is a sensitive and an absolute quantitative method, which can be used to detect HER2 CNV. Three HER2 exon-specific digital PCR assays along with three new reference genes assays (homo sapiens ribonuclease P RNA component H1 (RPPH1), glucose-6-phosphate isomerase (GPI), and chromosome 1 open reading frame 43 (C1ORF43), on different chromosomes) were established and validated by using standard reference material, 8 different cell lines and 110 clinical Formalin-fixed and paraffin-embedded (FFPE) samples. DPCR can achieve precise quantification of HER2 CNV by calculating the ratio of HER2/reference gene. The positive and negative coincidence rates were 98% (53/54) and 95% (53/56), respectively, compared with fluorescence in situ hybridization (FISH) diagnostic result 110 of FFPE samples. The common reference gene CEP17 used for FISH diagnostic was not suitable as single reference gene for HER2 CNV measurements by dPCR. The best practice of HER2 CNV determination by dPCR is to conduct the three duplex assays of H1 (HER2 exon 4) with the proposed three new reference genes, with a positive cut-off value of H1/RPPH1 ≥ 2.0 or H1/averaged reference gene ≥ 2.0. The proposed dPCR method in our study can accurately provide absolute copy number of HER2 and reference gene on an alternative chromosome, thus avoiding false negative caused by polysomy of chromosome 17. The improved molecular typing and diagnosis of breast cancer will better guide clinical medication.

Development of highly accurate digital PCR method and reference material for monkeypox virus detection.

Human monkeypox has attracted attention recently. Monkeypox virus (MPXV) keeps evolving as it spreading around the world rapidly, which may threaten the health of more and more people. Here, we have developed a high order reference method based on digital PCR (dPCR) for MPXV detection, of which the limits of quantification (LoQ) and detection (LoD) are 38 and 6 copies/reaction, respectively. Pseudovirus reference materials (RM) containing the conserved F3L gene has been developed, and the homogeneity assessment showed that the RM was homogeneous. The reference value with its expanded uncertainty determined by the established dPCR is (2.74 ± 0.46) × 103 copies/µL. Six different MPXV test kits were accessed by the RM. Four out of six test kits cannot reach their claimed LoDs. The poor analytical sensitivity might cause false-negative results, which lead to incorrect diagnosis and treatment. The establishment of a high order reference method of dPCR and pseudovirus RM is very useful for improving the accuracy and reliability of MPXV detection.

Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics.

Coronavirus disease 2019 (COVID-19) is an infectious, acute respiratory disease caused mainly by person-to-person transmission of the coronavirus SARS-CoV-2. Its emergence has caused a world-wide acute health crisis, intensified by the challenge of reliably identifying individuals likely to transmit the disease. Diagnosis is hampered by the many unknowns surrounding this disease, including those relating to infectious viral burden. This uncertainty is exacerbated by disagreement surrounding the clinical relevance of molecular testing using reverse transcription quantitative PCR (RT-qPCR) for the presence of viral RNA, most often based on the reporting of quantification cycles (Cq), which is also termed the cycle threshold (Ct) or crossing point (Cp). Despite it being common knowledge that Cqs are relative values varying according to a wide range of different parameters, there have been efforts to use them as though they were absolute units, with Cqs below an arbitrarily determined value, deemed to signify a positive result and those above, a negative one. Our results investigated the effects of a range of common variables on Cq values. These data include a detailed analysis of the effect of different carrier molecules on RNA extraction. The impact of sample matrix of buccal swabs and saliva on RNA extraction efficiency was demonstrated in RT-qPCR and the impact of potentially inhibiting compounds in urine along with bile salts were investigated in RT-digital PCR (RT-dPCR). The latter studies were performed such that the impact on the RT step could be separated from the PCR step. In this way, the RT was shown to be more susceptible to inhibitors than the PCR. Together, these studies demonstrate that the consequent variability of test results makes subjective Cq cut-off values unsuitable for the identification of infectious individuals. We also discuss the importance of using reliable control materials for accurate quantification and highlight the substantial role played by dPCR as a method for their development.

Application of an eDNA assay for the detection of Pseudoloma neurophilia (Microsporidia) in zebrafish (Danio rerio) facilities.

Environmental DNA (eDNA) water assays are beginning to be implemented for many important pathogens in confined aquaculture systems. Recirculating systems are rapidly being developed for fin fish aquaculture. Zebrafish (Danio rerio) are reared in these systems, and Pseudoloma neurophilia (Microsporidia) represents a serious challenge for zebrafish research facilities. Diagnosis of the pathogen has traditionally used histology or PCR of tissues with lethal sampling. However, with the development of a nonlethal assay to detect P. neurophilia in tank water, facilities will be able to integrate the assay into routine surveillance efforts to couple with their established protocols. Here, we first describe a modified protocol to extract and quantify parasite DNA from the environment for nonlethal detection of P. neurophilia in adult zebrafish populations. Using this modified assay, we then evaluated water samples from a longitudinal experimental infection study, targeting timepoints during initial infection. The parasite was detectable in the water immediately after initial exposure until week 4 post exposure (pe), when the parasite was undetectable until 7 weeks pe. After that time, the parasite was sporadically detected in the water for the 10-month study, likely correlating with the lifecycle of the parasite. Using water samples from the Zebrafish International Resource Center, we also validated the clinical relevance of the assay in a large zebrafish facility. The integration of this assay at ZIRC will significantly compliment surveillance and control efforts for the microsporidian parasite.

A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations.

Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequences from the wild-type sequence is limited by the probe's tolerance to mismatch. To address this, we have developed a novel dPCR method that uses a primer instead of a probe to sense gene variation. The enhanced Taq DNA polymerase in the PCR system has a high mismatch sensitivity, which enables our dPCR method to distinguish gene mutations from wild-type sequences. Compared to current dPCR methods, our method shows superior precision in assessing gene editing efficiency and single-base DNA mutation. This presents a promising opportunity to advance gene editing research and rare gene mutation detection.

Clinimetric properties of the diagnostic criteria for psychosomatic research among the elderly.

INTRODUCTION: Among the elderly, the availability of tool assessing psychosomatic syndromes is limited. The present study aims at testing inter-rater reliability and concurrent validity of the semi-structured interview for the Diagnostic Criteria for Psychosomatic Research (DCPR-R-SSI) in the elderly of the general population. METHOD: One hundred eight subjects were recruited. Participants received a clinical assessment which included the DCPR-R-SSI, the Illness Attitude Scale (IAS), the Geriatric Depression Scale (GDS), the Psychosocial Index (PSI), the Toronto Alexithymia Scale-20 (TAS-20). Analyses of inter-rater reliability of DCPR-R-SSI and concurrent validity between DCPR-R-SSI and self-administered questionnaires were conducted. RESULTS: DCPR-R-SSI showed excellent inter-rater reliability with a percent of agreement of 90.7% (K Cohen: 0.856 [SE = 0.043], 95% CI: 0.77-0.94). DCPR-R demoralization showed fair concurrent validity with GDS; concurrent validity was also fair between DCPR-R Alexithymia and TAS-20, and between DCPR-R allostatic overload and PSI allostatic load, while the concurrent validity between DCPR-R Disease Phobia and IAS was moderate. CONCLUSION: DCPR-R-SSI represents a reliable and valid tool to assess psychosomatic syndromes in the elderly. DCPR-R is in need of being implemented in the elderly clinical evaluation.

[Digital PCR as a Highly Sensitive Diagnostic Tool: A Review].

Digital PCR (dPCR) is a nucleic acid quantification method that is widely used in genetic analysis. One of the most significant advantages of dPCR over other methods is the possibility of absolute quantitative determination of genetic material without construction of calibration curves, which allows one to detect even single molecules of nucleic acids, and, hence, provides early diagnosis of diseases. One specific characteristic of dPCR is the detection of the analyzed biological object in each microreaction, followed by the presentation of the analysis results in a binary system, thereby giving the method its name. The key aspects of developing the dPCR method, i.e., from the first devices based on microfluidic chip technology to modern systems capable of measuring a target at a concentration of up to 1 in 100000 copies are shown in the current work. We analyzed the data on the detection of various pathogens using dPCR, as well as summarizing various study results demonstrating the innovativeness of this method. Both the possibilities of multiplex dPCR analysis and its potential in clinical practice are presented. This review also addresses the issue of the role of dPCR in the development of noninvasive methods for analysis of oncological diseases. Possible ways of developing dPCR technology were emphasized, including its use as a "point-of-care" system.

Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach.

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.

Near-Infrared Responsive Droplet for Digital PCR.

Digital PCR (dPCR) surpasses the performance of earlier PCR formats because of highly precise, absolute quantification and other unique merits. A simple thermocycling approach and durable microcarrier are of great value for dPCR advancement and application. Herein, a near-infrared (NIR) controlled thermocycling approach by embedding magnetic graphene oxide (GO) composite into the agarose microcarriers is developed. The core-shell composite is constructed by sequentially encapsulating GO and silica outside the magnetic nanocores. Benefiting from these additives, the resultant composite agarose gains appealing features as light-driven temperature changing, switchable gel-sol phase transforming, biocompatibility, and magnetic traction. By further emulsifying into droplets via the microfluidics method, the influence of typical parameters including material loading amount, laser intensity, and droplet diameter at various ranges is investigated for assembling microcarriers with different responsiveness. Then a paradigm of the NIR program can be easily tailored for PCR thermocycling. Finally, the feasibility of the approach is verified by detecting statistically diluted Klebsiella pneumoniae DNA samples, from 0.1 to 2 copies per drop. It is anticipated that this method has promising prospects for dPCR-based and other temperature-controlled applications.