PCR Techniques and Their Clinical Applications

Kary B. Mullis developed a revolutionary method name polymerase chain reaction (PCR) in 1983, which can synthesize new strand of DNA complementary to the template strand of DNA and produce billions of copies of a DNA fragment only in few hours. Denaturation, annealing, and extension are the three primary steps involved in the PCR process, which generally requires thermocyclers, DNA template, a pair of primers, Taq polymerase, nucleotides, buffers, etc. With the development of PCR, from traditional PCR, quantitative PCR, to next digital PCR, PCR has become a powerful tool in life sciences and medicine. Applications of PCR techniques for infectious diseases include specific or broad-spectrum pathogen detection, assessment and surveillance of emerging infections, early detection of biological threat agents, and antimicrobial resistance analysis. Applications of PCR techniques for genetic diseases include prenatal diagnosis and screening of neonatal genetic diseases. Applications of PCR techniques for cancer research include tumor-related gene detection. This chapter aimed to discuss about the different types of PCR techniques, including traditional PCR, quantitative PCR, digital PCR, etc., and their applications for rapid detection, mutation screen or diagnosis in infectious diseases, inherited diseases, cancer, and other diseases

The influence of summer hypoxia on sedimentary phosphorus biogeochemistry in a coastal scallop farming area, North Yellow Sea

In situ field investigations coupled with laboratory incubations were employed to explore the surface sedimentary phosphorus (P) cycle in a mariculture area adjacent to the Yangma Island suffering from summer hypoxia in the North Yellow Sea. Five forms of P were fractionated, namely exchangeable P (Ex-P), iron-bound P (Fe-P), authigenic apatite (Ca-P), detrital P (De-P) and organic P (OP). Total P (TP) varied from 13.42 to 23.88 mu mol g(-1) with the main form of inorganic P (IP). The benthic phosphate (DIP) fluxes were calculated based on incubation experiments. The results show that the sediment was an important source of P in summer with similar to 39% of the bioavailable P (Bio-P) recycled back into the water column. However, the sediment acted a sink of P in autumn. The benthic DIP fluxes were mainly controlled by the remobilizing of Fe-P, Ex-P and OP under contrasting redox conditions. In August (hypoxia season), similar to 0.92 mu mol g(-1) of Fe-P and similar to 0.52 mu mol g(-1) of OP could be transformed to DIP and released into water, while similar to 0.36 mu mol g(-1) of DIP was adsorbed to clay minerals. In November (non-hypoxia season), however, similar to 0.54 mu mol g(-1) of OP was converted into DIP, while similar to 0.55 mu mol g(-1) and similar to 0.28 mu mol g(-1) of DIP was adsorbed to clay minerals and bind to iron oxides. Furthermore, scallop farming activities also affected the P mobilization through biological deposition and reduced hydrodynamic conditions. The burial fluxes of P varied from 11.67 to 20.78 mu mol cm(-2) yr(-1) and its burial efficiency was 84.7-100%, which was consistent with that in most of the marginal seas worldwide. This study reveals that hypoxia and scallop farming activities can significantly promote sedimentary P mobility, thereby causing high benthic DIP flux in coastal waters. (C) 2020 Elsevier B.V. All rights reserved

Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

10.1371/journal.pone.0074040PLoS ONE89-POLN

Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma.

BACKGROUND: Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Gene-expression profiling has identified subgroups of DLBCL (activated B-cell-like [ABC], germinal-center B-cell-like [GCB], and unclassified) according to cell of origin that are associated with a differential response to chemotherapy and targeted agents. We sought to extend these findings by identifying genetic subtypes of DLBCL based on shared genomic abnormalities and to uncover therapeutic vulnerabilities based on tumor genetics. METHODS: We studied 574 DLBCL biopsy samples using exome and transcriptome sequencing, array-based DNA copy-number analysis, and targeted amplicon resequencing of 372 genes to identify genes with recurrent aberrations. We developed and implemented an algorithm to discover genetic subtypes based on the co-occurrence of genetic alterations. RESULTS: We identified four prominent genetic subtypes in DLBCL, termed MCD (based on the co-occurrence of MYD88L265P and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Genetic aberrations in multiple genes distinguished each genetic subtype from other DLBCLs. These subtypes differed phenotypically, as judged by differences in gene-expression signatures and responses to immunochemotherapy, with favorable survival in the BN2 and EZB subtypes and inferior outcomes in the MCD and N1 subtypes. Analysis of genetic pathways suggested that MCD and BN2 DLBCLs rely on "chronic active" B-cell receptor signaling that is amenable to therapeutic inhibition. CONCLUSIONS: We uncovered genetic subtypes of DLBCL with distinct genotypic, epigenetic, and clinical characteristics, providing a potential nosology for precision-medicine strategies in DLBCL. (Funded by the Intramural Research Program of the National Institutes of Health and others.).This research was supported by the Intramural Research Program of the NIH, Center for Cancer Research, National Cancer Institute and by a National Cancer Institute Strategic Partnering to Evaluate Cancer Signatures (SPECS II) grant (5U01CA157581-05). R.S. was supported by the Dr Mildred Scheel Stiftung für Krebsforschung (Deutsche Krebshilfe). D.J.H. was a Kay Kendall Leukaemia Fund Intermediate research fellow. M.K. was supported by the National Institutes of Health Oxford-Cambridge Scholars Program and the Washington University in St. Louis Medical Scientist Training Progra

AtSK11 and AtSK12 Mediate the Mild Osmotic Stress-Induced Root Growth Response in Arabidopsis

Although most osmotic stresses are harmful to plant growth and development, certain drought- or polyethylene glycol (PEG)-induced mild osmotic stresses promote plant root growth. The underlying regulatory mechanisms of this response remain elusive. Here, we report that the GLYCOGEN SYNTHASE KINASE 3 (GSK3) genes ARABIDOPSIS THALIANA SHAGGY-RELATED KINASE 11 (AtSK11) (AT5G26751) and AtSK12 (AT3G05840) are involved in the mild osmotic stress (−0.4 MPa) response in Arabidopsis thaliana. When grown on plant medium infused with different concentrations of PEG to mimic osmotic stress, both wild-type (WT) and atsk11atsk12 plants showed stimulated root growth under mild osmotic stress (−0.4 MPa) but repressed root growth under relatively strong osmotic stress (−0.5, −0.6, −0.7 MPa) as compared to the mock condition (−0.25 MPa). The root growth stimulation of atsk11atsk12 was more sensitive to −0.4 MPa treatment than was that of WT, indicating that AtSK11 and AtSK12 inhibit the mild stress-induced root growth response. RNA-seq analysis of WT and atsk11atsk12 plants under three water potentials (−0.25 MPa, −0.4 MPa, −0.6 MPa) revealed 10 differentially expressed candidate genes mainly involved in cell wall homeostasis, which were regulated by AtSK11 and AtSK12 to regulate root growth in response to the mild stress condition (−0.4 MPa). Promoter motif and transcription factor binding analyses suggested that the basic helix-loop-helix (bHLH) transcription factor bHLH69/LJRHL1-LIKE 2 (LRL2) may directly regulate the expression of most −0.4 MPa-responsive genes. These findings indicate that mild osmotic stress (−0.4 MPa) promotes plant growth and that the GSK3 family kinase genes AtSK11 and AtSK12 play a negative role in the induction of root growth in response to mild osmotic stress

Adsorption kinetics of platinum group elements onto macromolecular organic matter in seawater

Adsorption kinetics of the interaction between Pt, Pd and Rh (defined here as platinum group elements, PGEs) ions and macromolecular organic compounds (MOCs, >10 kDa), including humic acid, carrageenan and bovine serum albumin, and different cutoff fractions of natural organic matter (>1 kDa and >3 kDa) obtained from seawater using centrifugal ultrafiltration devices were investigated. For a given element, all the adsorption kinetics did not reach equilibrium except the interaction between Pt and >1 kDa cutoff, and between Pd and humic acid. For all the tested MOCs, the adsorption kinetics could be divided into two stages, a rapid adsorption process in the first 8 h and the desorption stage after the first 8 h until the equilibrium. The change trend of partition coefficient (log_(10)K_d) values with experiment time was consistent with that of the kinetic curves. However, in the interaction between PGE ions and natural dissolved organic matter (NDOM), an obvious difference in the change trends of log_(10)K_d and kinetic curves was observed. It indicated that the partition behavior of PGE ions interacting with NDOM in seawater was a combined effect of different organic constituents. The adsorption and log_(10)K_d of PGEs in the >1 kDa NDOM fraction were higher and more stable than those in the >3 kDa NDOM fraction. The results also indicated that the 1–3 kDa NDOM may dominate the interaction between PGEs ions and NDOM. Moreover, no kinetic model could perfectly simulate the adsorption process. It indicated that the colloidal struction and morphology of MOCs or NDOM in seawater might be inhomogeneous. Hence, the interaction between PGE ions and organic matter in seawater was a complicated process and needs further research