Preparation of Labeled DNA, RNA, and Oligonucleotide Probes

Labeled nucleic acids and oligonucleotides are typically generated by enzymatic methods such as end-labeling, random priming, nick translation, in vitro transcription, and variations of the polymerase chain reaction (PCR). Some of these methods place the label in specific locations within the nucleic acid (e.g., at the 5′ or 3′ terminus); others generate molecules that are labeled internally at multiple sites. Some methods yield labeled single-stranded products, whereas others generate double-stranded nucleic acids. Finally, some generate probes of defined length, whereas others yield a heterogeneous population of labeled molecules. Options available for generating and detecting labeled nucleic acids, as well as advice on designing oligonucleotides for use as probes, is included here.

Enzymatic Methods for Salivary Biomarkers Detection: Overview and Current Challenges

Early detection is a key factor in patient fate. Currently, multiple biomolecules have been recognized as biomarkers. Nevertheless, their identification is only the starting line on the way to their implementation in disease diagnosis. Although blood is the biofluid par excellence for the quantification of biomarkers, its extraction is uncomfortable and painful for many patients. In this sense, there is a gap in which saliva emerges as a non-invasive and valuable source of information, as it contains many of the biomarkers found in blood. Recent technological advances have made it possible to detect and quantify biomarkers in saliva samples. However, there are opportunity areas in terms of cost and complexity, which could be solved using simpler methodologies such as those based on enzymes. Many reviews have focused on presenting the state-of-the-art in identifying biomarkers in saliva samples. However, just a few of them provide critical analysis of technical elements for biomarker quantification in enzymatic methods for large-scale clinical applications. Thus, this review proposes enzymatic assays as a cost-effective alternative to overcome the limitations of current methods for the quantification of biomarkers in saliva, highlighting the technical and operational considerations necessary for sampling, method development, optimization, and validation.

Cryptococcus neoformans capsule regrowth experiments reveal dynamics of enlargement and architecture

The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded characterization. Polysaccharides remain attached to the cell as capsular polysaccharide (CPS) or are shed into the surroundings in the form of exopolysaccharide (EPS). While a great deal of study has been done examining the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and removed capsular material. Sonication and glucanex digestion yield soluble CPS preparations, while French Press and modified glucanex digestion plus vortexing remove the capsule and cell wall producing polysaccharide aggregates that we call capsule ghosts. The existence of capsule ghosts implies an inherent organization that allows it to exist independent of the cell wall surface. As sonication and glucanex digestion were noncytotoxic, it was possible to observe the cryptococcal cells rebuilding their capsule, revealing new insights into capsule architecture and synthesis consistent with a model in which the capsule is assembled from smaller polymers, which are then assemble into larger ones.

KESESUAIAN KOLESTEROL LDL HASIL PERHITUNGAN SEJUMLAH FORMULA DENGAN KOLESTEROL LDL DIREK METODE ENZIMATIK

Increased cholesterol levels are the main cause of coronary heart disease. The results of examining LDL must be precise and accurate, but direct LDL examination (LDL-Direct) is quite expensive to do in a place with limited facilities so various experiments are carried out to get LDL formula (LDL-Cal) that is appropriate than formula commonly used, Friedewald formula. The aimed of this study was to determine the correlation between LDL cholesterol from calculation a number formulas with direct LDL cholesterol in order to obtain the best formula to be applied in laboratory of Lubuk Sikaping Hospital. This research was conducted on 75 patients who did lipid profile examination in laboratory of Lubuk Sikaping Hospital, who meet the inclusion criteria. Examination of total cholesterol, HDL, LDL and triglycerides were carried out by enzymatic methods on clinical chemistry analyzer. LDL cholesterol also calculated by several formulas namely Friedelwald formula (TC- (TG/5) -HDL), Chen formula (90% nonHDLC-10% TG), Anandaraja formula (0.9 TC- (0.9 TG/5) -28), Puavilai formula (TC-HDLC-TG/6), Vujovic formula (TC-HDLC-TG/3), and Cordova formula (3/4 (TC-HDLC.) Bland & Altman plot was used to compare the calculated LDL cholesterol level of each formula with the direct LDL. The mean LDL levels were 136.41 (35.92); 116.64 (32.72); 117.03 (30.83); 121.95 (32.79); 121.83 (33.23); 96.84 (32.47); 109.97 (28.24) for Direct, Friedelwald, Chen, Anandaraja, Puavilai, Vujovic, and Cordova respectively. Based on the Bland & Altman Plot, the calculation of LDL cholesterol from the Chen formula has the best compatibility with LDL-Direk with the mean difference of 19.3867 ± 19.0489 mg / dl at triglyceride levels <400 mg / dl, so it can be applied at RSUD Lubuk Sikaping with limited facilities. Keywords: LDL Cholesterol; LDL-Direct; LDL-CalAbstrakPeningkatan kadar kolesterol merupakan penyebab utama penyakit jantung koroner. Hasil pemeriksaan kadar LDL serum harus tepat dan akurat, namun pemeriksaan kadar LDL secara langsung (LDL-Direk) cukup mahal untuk dilakukan di tempat dengan fasilitas terbatas sehingga dilakukan berbagai percobaan untuk mendapatkan formula perhitungan LDL (LDL-Cal) yang lebih tepat dibandingkan formula yang telah umum digunakan yaitu formula Friedewald. Penelitian ini bertujuan untuk mengetahui kesesuaian antara kolesterol LDL hasil perhitungan sejumlah formula dengan kolesterol LDL direk sehingga diperoleh formula perhitungan terbaik untuk dapat diterapkan di laboratorium RSUD Lubuk Sikaping. Penelitian ini dilakukan terhadap 75 orang pasien yang melakukan pemeriksaan profil lipid lengkap ke Laboratorium RSUD Lubuk Sikaping. yang memenuhi kriteria inklusi. Pemeriksaan kolesterol total, HDL, LDL dan trigliserida dilakukan dengan metode enzimatik pada alat kimia klinik otomatis. Untuk kolesterol LDL juga dihitung dengan beberapa rumus yaitu formula Friedelwald (TC-(TG/5)-HDL). formula Chen (90%nonHDLC-10%TG), formula Anandaraja (0.9 TC- (0.9 TG/5)-28), formula Puavilai (TC-HDLC-TG/6), formula Vujovic (TC-HDLC-TG/3), dan formula Cordova (3/4 (TC-HDLC). Bland & Altman plot digunakan untuk membandingkan antara kadar kolesterol LDL hasil hitung masing-masing formula dengan LDL direk. Rerata kadar LDL (mg/dl) berturut-turut adalah 136,41 (35,92); 116,64 (32,72); 117,03 (30,83); 121,95 (32,79); 121,83 (33,23); 96,84 (32,47); 109,97 (28,24) untuk LDL-Direk, Friedelwald, Chen, Anandaraja, Puavilai, Vujovic, dan Cordova. Berdasarkan Bland & Altman Plot, perhitungan kolesterol LDL Formula Chen memiliki kesesuaian paling baik dengan LDL-Direk dengan selisih rerata 19,3867 ± 19,0489 mg/dl pada kadar trigliserida < 400 mg/dl, sehingga dapat diterapkan di RSUD Lubuk Sikaping dengan fasilitas yang terbatas.

Progress and challenges in the synthesis of sequence controlled polysaccharides

The sequence, length and substitution of a polysaccharide influence its physical and biological properties. Thus, sequence controlled polysaccharides are important targets to establish structure–properties correlations. Polymerization techniques and enzymatic methods have been optimized to obtain samples with well-defined substitution patterns and narrow molecular weight distribution. Chemical synthesis has granted access to polysaccharides with full control over the length. Here, we review the progress towards the synthesis of well-defined polysaccharides. For each class of polysaccharides, we discuss the available synthetic approaches and their current limitations.

Large-Scale de novo Oligonucleotide Synthesis for Whole-Genome Synthesis and Data Storage: Challenges and Opportunities

Over the past decades, remarkable progress on phosphoramidite chemistry-based large-scale de novo oligonucleotide synthesis has been achieved, enabling numerous novel and exciting applications. Among them, de novo genome synthesis and DNA data storage are striking. However, to make these two applications more practical, the synthesis length, speed, cost, and throughput require vast improvements, which is a challenge to be met by the phosphoramidite chemistry. Harnessing the power of enzymes, the recently emerged enzymatic methods provide a competitive route to overcome this challenge. In this review, we first summarize the status of large-scale oligonucleotide synthesis technologies including the basic methodology and large-scale synthesis approaches, with special focus on the emerging enzymatic methods. Afterward, we discuss the opportunities and challenges of large-scale oligonucleotide synthesis on de novo genome synthesis and DNA data storage respectively.

Enzymatic Methods for the Site-Specific Radiolabeling of Targeting Proteins

Site-specific conjugation of proteins is currently required to produce homogenous derivatives for medicine applications. Proteins derivatized at specific positions of the polypeptide chain can actually show higher stability, superior pharmacokinetics, and activity in vivo, as compared with conjugates modified at heterogeneous sites. Moreover, they can be better characterized regarding the composition of the derivatization sites as well as the conformational and activity properties. To this aim, several site-specific derivatization approaches have been developed. Among these, enzymes are powerful tools that efficiently allow the generation of homogenous protein–drug conjugates under physiological conditions, thus preserving their native structure and activity. This review will summarize the progress made over the last decade on the use of enzymatic-based methodologies for the production of site-specific labeled immunoconjugates of interest for nuclear medicine. Enzymes used in this field, including microbial transglutaminase, sortase, galactosyltransferase, and lipoic acid ligase, will be overviewed and their recent applications in the radiopharmaceutical field will be described. Since nuclear medicine can benefit greatly from the production of homogenous derivatives, we hope that this review will aid the use of enzymes for the development of better radio-conjugates for diagnostic and therapeutic purposes.