High DNA Concentrations Research Articles

Robustness of digital PCR and real‐time PCR against inhibitors in transgene detection for gene doping control in equestrian sports

Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time PCR against gene doping. Digital PCR amplification was significantly inhibited by high concentrations of proteinase K (more than 0.1μg/μl), ethylenediaminetetraacetic acid (more than 5nmol/μl), and heparin (more than 0.05 unit/μl) but not by ethanol or genomic DNA. In addition, phenol affected droplet formation in the digital PCR amplification process. Real-time PCR amplification was inhibited by high concentrations of phenol (more than 1% v/v), proteinase K (more than 0.001 μg/μl), ethylenediaminetetraacetic acid (more than 1nmol/μl), heparin (more than 0.005 unit/μl), and genomic DNA (more than 51.9ng/μl) but not by ethanol. Although both PCR systems were inhibited by nearly the same substances, digital PCR was more robust than real-time PCR against the inhibitors. We believe that our findings are important for the development of better methods for transgene detection and prevention of false negative results in gene doping testing.

DNA isolation of clove and lemongrass using modification of the Doyle and Doyle methods and their relation with antioxidant activity

Clove (Syzigium aromaticum) and lemongrass (Cymbopogon winterianus) are essential oil-producing plants that are widely available in Indonesia and have high economic value for medicinal and industrial needs because of their antioxidant activity. However, the information about the relationship between antioxidant activity and the DNA content in these plants was very limited. Especially because DNA isolation of aromatic plants is also not easy to do with methods that are widely used. Therefore, this study aims to isolate DNA from clove and lemongrass using a modified Doyle and Doyle method and to find the relationship between the quality and quantity of DNA with their antioxidant activity. The method used was the use of PVP, isopropanol and sodium acetate for the modification of the Doyle and Doyle method in DNA isolation followed by antioxidant analysis using DPPH. The results showed that the modification to the Doyle and Doyle method had obtained high concentrations of DNA. Besides that, the clove DNA content is higher than lemongrass which is supported by the antioxidant activity of both plants.

DNA Minor Groove-Induced cis-trans Isomerization of a Near-Infrared Fluorescent Probe.

The discovery of small molecules that exhibit turn-on far-red or near-infrared (NIR) fluorescence upon DNA binding and understanding how they bind DNA are important for imaging and bioanalytical applications. Here we report the DNA-bound structure and the DNA binding mechanism of quinone cyanine dithiazole (QCy-DT), a recently reported AT-specific turn-on NIR fluorescent probe for double-stranded DNA. The nuclear magnetic resonance (NMR)-derived structure showed minor groove binding but no specific ligand-DNA interactions, consistent with an endothermic and entropy-driven binding mechanism deduced from isothermal titration calorimetry. Minor groove binding is typically fast because it minimally perturbs the DNA structure. However, QCy-DT exhibited unusually slow DNA binding. The cyanine-based probe is capable of cis-trans isomerization due to overlapping methine bridges, with 16 possible slowly interconverting cis/trans isomers. Using NMR, density functional theory, and free energy calculations, we show that the DNA-free and DNA-bound environments of QCy-DT prefer distinctly different isomers, indicating that the origin of the slow kinetics is a cis-trans isomerization and that the minor groove preferentially selects an otherwise unstable cis/trans isomer of QCy-DT. Flux analysis showed the conformational selection pathway to be the dominating DNA binding mechanism at low DNA concentrations, which switches to the induced fit pathway at high DNA concentrations. This report of cis/trans isomerization of a ligand, upon binding the DNA minor groove, expands the prevailing understanding of unique discriminatory powers of the minor groove and has an important bearing on using polymethine cyanine dyes to probe the kinetics of molecular interactions.

Tailoring the growth and proliferation of human dermal fibroblasts by DNA-based polymer films for skin regeneration.

The use of DNA as a functional biomaterial for therapeutic, diagnostic, and drug delivery applications has been prominent in recent years, but its use as a scaffold for tissue regeneration is still limited. This study aimed to evaluate the biocompatibility and interaction of DNA-based polymeric films (DNA-PFs) with primary human fibroblasts (PHF) for regenerative medicine and wound healing purposes. The morphological characterization of the films was performed by scanning electron microscopy, SEM-energy-dispersive X-ray spectroscopy, and atomic force microscopy analysis. Cell viability, cell cycle kinetics, oxidative stress, and migration studies were carried out at 48 and 72 hr of incubation and compared to control cells. Cell adhesion was impaired in the first 24 hr, DNA-PFs with higher concentrations of DNA (1.0 and 2.0g/L) this effect was not seen in DNA-PFs (0.5g/L), explained by the difference in topography and roughness of DNA-PFs, but it was overcome after 48 hr of incubation. PHF seeded on DNA films showed higher proliferation and migration rates than the control after 48 hr of incubation, with the maintenance of cell morphology and lower cytotoxicity and oxidative stress during the evaluation time. Therefore, these results indicate that DNA-PFs are highly biocompatible and provide a suitable microenvironment for dermal fibroblasts to maintain their activity, helping build new and more complex biomaterials suitable for future tissue repair applications.

Unraveling the salt induced modulation in the photophysical behavior of acridine orange dye on its interaction with natural DNA

Present study explores the interaction of biologically important dye, acridine orange (AOH+), with biomacromolecular host, DNA, under two extreme salt (NaCl) concentration conditions, i. e., physiologically relevant 0.15 M NaCl and highly concentrated 4 M NaCl, following various photophysical measurements. In the presence of the salt, the AOH+-DNA system shows contrasting modulations in the photophysical properties than those reported earlier in the absence of the salt. Thus, without any salt addition, the AOH+ undergoes a drastic fluorescence quenching at the lower DNA concentrations, due to dye dimerization on DNA surface, but experiences a huge fluorescence enhancement at higher DNA concentrations, owing to monomeric dye-DNA intercalative complex formation. Contrastingly, in the presence of the salt, we observe only large fluorescence enhancement for AOH+ all along the DNA concentrations used. Critical inspection of the results reveal that at 0.15 M NaCl, the monomeric form of AOH+ undergoes both DNA surface binding and intercalation between DNA base pairs. At 4 M NaCl, however, there is mostly the intercalative binding of AOH+ with DNA host. Interestingly, in the fluorescence anisotropy study, we observe the intriguing dip-rise-dip features in the anisotropy kinetic traces, rationalized considering dynamic variations in the fluorescence intensity and anisotropy contributions of the free and the DNA-bound AOH+ species present in the solution. Present study provides the details of the salt-induced modulations in the photophysical properties of AOH+ upon its interaction with DNA host, thereby revealing the intriguing insights of the binding mechanisms involved in the present systems. Observed results would expectedly be useful in bio-analytical chemistry, pharmaceuticals, drug formulations, drug delivery, functional materials, and similar other applications.

Microbiome-based environmental monitoring of a dairy processing facility highlights the challenges associated with low microbial-load samples

Efficient and accurate identification of microorganisms throughout the food chain can potentially allow the identification of sources of contamination and the timely implementation of control measures. High throughput DNA sequencing represents a potential means through which microbial monitoring can be enhanced. While Illumina sequencing platforms are most typically used, newer portable platforms, such as the Oxford Nanopore Technologies (ONT) MinION, offer the potential for rapid analysis of food chain microbiomes. Initial assessment of the ability of rapid MinION-based sequencing to identify microbes within a simple mock metagenomic mixture is performed. Subsequently, we compare the performance of both ONT and Illumina sequencing for environmental monitoring of an active food processing facility. Overall, ONT MinION sequencing provides accurate classification to species level, comparable to Illumina-derived outputs. However, while the MinION-based approach provides a means of easy library preparations and portability, the high concentrations of DNA needed is a limiting factor.

A minimally destructive protocol for DNA extraction from ancient teeth.

Ancient DNA sampling methods—although optimized for efficient DNA extraction—are destructive, relying on drilling or cutting and powdering (parts of) bones and teeth. As the field of ancient DNA has grown, so have concerns about the impact of destructive sampling of the skeletal remains from which ancient DNA is obtained. Due to a particularly high concentration of endogenous DNA, the cementum of tooth roots is often targeted for ancient DNA sampling, but destructive sampling methods of the cementum often result in the loss of at least one entire root. Here, we present a minimally destructive method for extracting ancient DNA from dental cementum present on the surface of tooth roots. This method does not require destructive drilling or grinding, and, following extraction, the tooth remains safe to handle and suitable for most morphological studies, as well as other biochemical studies, such as radiocarbon dating. We extracted and sequenced ancient DNA from 30 teeth (and nine corresponding petrous bones) using this minimally destructive extraction method in addition to a typical tooth sampling method. We find that the minimally destructive method can provide ancient DNA that is of comparable quality to extracts produced from teeth that have undergone destructive sampling processes. Further, we find that a rigorous cleaning of the tooth surface combining diluted bleach and UV light irradiation seems sufficient to minimize external contaminants usually removed through the physical removal of a superficial layer when sampling through regular powdering methods.

Self-Assembled DNA Nanoflowers Triggered by a DNA Walker for Highly Sensitive Electrochemical Detection of Staphylococcus aureus.

With the development of DNA nanotechnology, DNA has been widely used to construct a variety of nanomachines. Among them, a DNA walker is a unique nanomachine that can move continuously along a specific orbit to fulfill diverse functions. In this paper, a dual signal amplification electrochemical biosensor based on a DNA walker and DNA nanoflowers is constructed for high sensitivity detection of Staphylococcus aureus (S.aureus). Two groups of double-stranded DNA are modified on the surface of a gold electrode. The binding of S.aureus with its aptamer induces the disintegration of the long double strands and releases the DNA walker. With the help of exonuclease III (Exo III), the DNA walker moves along the electrode surface and continuously hydrolyzes the anchored short double strands. The introduction of a specially customized circular DNA and phi29 DNA polymerase initiates the rolling circle amplification (RCA) reaction. DNA nanoflowers are formed at high local concentration of DNA in the solution, which provide binding sites for electroactive methylene blue (MB) and thus produce intense signal. Under the best conditions, the current response is linearly related to the logarithm of the concentration of S.aureus ranging from 60 to 6 × 107 CFU/mL, and the detection limit is 9 CFU/mL. In addition, the proposed biosensor has achieved satisfactory results in the detection of actual water samples and diluted honey samples, which confirm the practicability of the biosensor and its application potential in environmental monitoring and food safety.

Comparison of Blood Bacterial Communities in Periodontal Health and Periodontal Disease.

The use of Next Generation Sequencing (NGS) techniques has generated a wide variety of blood microbiome data. Due to the large variation in bacterial DNA profiles between studies and the likely high concentrations of cell-free bacterial DNA in the blood, it is still not clear how such microbiome data relates to viable microbiota. For these reasons much remains to be understood about the true nature of any possible healthy blood microbiota and of bacteraemic events associated with disease. The gut, reproductive tracts, skin, and oral cavity are all likely sources of blood-borne bacteria. Oral bacteria, especially those associated with periodontal diseases, are also commonly associated with cardiovascular diseases such as infective endocarditis, and also have been linked to rheumatoid arthritis and Alzheimer’s disease. Periodontal treatment, dental probing, and toothbrushing have been shown to cause transient bacteraemia and oral bacteria from the phyla Firmicutes (e.g. Streptococci) and Bacteroidetes (e.g. Porphyromonas) are found in cardiovascular lesions (CVD). Many studies of blood bacterial DNA content however, find Proteobacteria DNA to be the dominant microbiome component, suggesting a gut origin. Most studies of this type use total DNA extracted from either whole blood or blood fractions, such as buffy coat. Here, using a method that purifies DNA from intact bacterial cells only, we examined blood donated by those with active, severe periodontitis and periodontally healthy controls and show that 43–52% of bacterial species in blood are classified as oral. Firmicutes, consisting largely of members of the Streptococcus mitis group and Staphylococcus epidermidis, were predominant at 63.5% of all bacterial sequences detected in periodontal health and, little changed at 66.7% in periodontitis. Compared to studies using total DNA Proteobacteria were found here at relatively low levels in blood at 13.3% in periodontitis and 17.6% in health. This study reveals significant phylogenetic differences in blood bacterial population profiles when comparing periodontal health to periodontal disease cohorts.

Value of the toluidine blue test as an aid to determine the biopsy site in actinic prurigo cheilitis

ABSTRACTActinic prurigo (AP) is a type of photodermatosis that primarily affects the Latin American mestizo population. Histologically, AP cheilitis exhibits acanthosis with spongiosis and vacuolation of the basal cell layer overlying a dense lymphocytic inflammatory infiltrate that forms well-defined lymphoid follicles. Toluidine blue is a thiazide, acidophilic, and metachromatic dye used in vivo to selectively stain the acidic components of tissues such as sulfates, carboxylates, and phosphate radicals that are incorporated into DNA and RNA. It is necessary to develop a method that allows detecting, on clinical grounds the area of the lesion in which it is more feasible to find such structures. Thus to increase the sensitivity of the biopsy, in AP cheilitis to accurately identify where the lymphoid follicles reside, based on the higher concentration of DNA in such structures and thus confirm the diagnosis. In this study, staining was positive in 85% of patients with AP cheilitis, in 14 of whom 82% lymphoid follicles were observed by histopathology. One of the pathologist’s problems in establishing the diagnosis of AP is that the main histopathological characteristics are not always identified in the submitted samples because it is not easy to clinically identify the most representative site of the lesion selected for performing a biopsy. Based on our results, we propose using toluidine blue as an auxiliary method to choose a tissue sample to facilitate the diagnosis and allow clinicians to make clinical correlations between the histopathological and therapeutic findings.

Specific distribution of human papillomavirus type in patients of different reproductive age with low and high-grade squamous intraepithelial lesion

The purpose of this work was to study the characteristics of human papillomaviruses spread in reproductive age patients with low and high degree squamous intraepithelial lesions of the cervix in Yekaterinburg. Material and Methods. In with study 140 female patients of reproductive age with histologically confirmed low and high grade intraepithelial lesions were retrospectively included. HPV-genotyping by polymerase chain reaction was carried out in combination with liquid-based cytology BDSurePath. Formed 4 groups: LSIL, age from 18 to 30 years; LSIL, age from 31 to 44; HSIL, ages 18 to 30; HSIL, ages 31 to 44. Results. Analysis of the obtained data demonstrated that HPV 16 prevails in intraepithelial squamous cell lesions of the cervix of low and high degree in women of reproductive age in Yekaterinburg. In women aged 18-30 years, compared with the older reproductive age (31-44 years), multiple infections with 3, 4 or more HPV genotypes are more often recorded, regardless of the degree of damage. In addition to HPV 16, low-grade lesions in young women are most often associated with HPV 31, 51, and 52, and high-grade lesions with types 31, 33 and 52. HPV 33 is characteristic of a high degree of intraepithelial squamous cell lesions, especially in patients of older reproductive age. With intraepithelial squamous lesions of low and high degree, regardless of age, an equally high concentration of HPV DNA is observed.

Population dynamics of Rhizoctonia, Oculimacula, and Microdochium species in soil, roots, and stems of English wheat crops

AbstractThis study aimed to elucidate the population dynamics of Rhizoctonia, Oculimacula, and Microdochium species, causing the stem base disease complex of sharp eyespot, eyespot, and brown foot rot in cereals. Pathogen DNA in soil, roots, and stem fractions, and disease expression were quantified in 102 English wheat fields in two seasons. Weather data for each site was collected to determine patterns that correlate with assessed diseases. Oculimacula spp. (66%) and R. solani AG 2‐1 (63%) were most frequently detected in soil, followed by R. cerealis (54%) and Microdochium spp. (33%). Oculimacula spp. (89%) and R. cerealis (56%) predominated on roots and soil but were not associated with root rot symptoms, suggesting that these species used soil and roots for survival and as inoculum source. M. nivale was more frequently detected than M. majus on stems up to GS 21–30 and co‐occurred on plant samples with O. acuformis. O. yallundae had higher DNA concentration than O. acuformis at the lower 5 cm basal region at GS 37–45. R. cerealis predominated in the upper 15 cm above the base beyond stem extension. Brown foot rot by Microdochium spp. was favoured by cool and wet autumns/winters and dominated in English wheat. Eyespot and sharp eyespot disease index by Oculimacula spp. and R. cerealis, respectively, correlated with wet/humid springs and summers. Results suggested that stem base pathogens generally coexisted; however, their abundance in time and space was influenced by favourable weather patterns and host development, with niche differentiation after stem extension.

PSV-36 Optimization of Collection, Storage, and Extraction of Samples for Large-Scale 16s Sequencing Project

Abstract Correlations between the microbiome of the horse and various physiological parameters continue to be determined. Most evaluations of the equine microbiome have been regionally focused with limited numbers or variability. In order to elucidate the role of the microbiome on equine health, a large-scale trial has been initiated with a primary objective of analyzing and characterizing the microbiome of the horse. A study of this scope relies on the horse owner to collect samples from various locations and ship them to a centralized lab. Therefore, collection protocols and extraction techniques need to be optimized to maintain sample integrity. A trial was conducted to evaluate the storage conditions and extraction methodologies necessary to develop an optimized method of sample collection, preparation, and extraction. Samples were collected from a single horse and subjected to one of four treatments in duplicate (1-fresh, 2-frozen, 3-stored in 99% EtOH, and 4-stored in a commercially available DNA/RNA transport medium; DNA/RNA Shield®, Zymo Research, Irvine, CA). Frozen samples were placed at -80◦ C immediately following collection and stored for 48-hr. Frozen samples were thawed immediately prior to extraction. All other treatments were stored as dictated by treatment at room temperature for 48-hr prior to extraction to mimic shipping. Samples were processed utilizing a commercially available DNA extraction kit (Quick-DNA™ Fecal/Soil Microbe Miniprep, Zymo Research). DNA extract was analyzed for quantity utilizing a Qubit Fluorometer (ThermoFisher, Waltham, MA), and quality via spectrophotometry (NanoDrop 2000; ThermoFisher, Waltham, MA). Swabs stored in DNA/RNA Shield® yielded higher concentrations of DNA with superior 260/280 values than other treatments. These data indicate that storage of fecal swabs in an appropriate buffer combined with optimized extraction protocols can deliver adequate extracts that can be utilized for 16s sequencing and validates the use of this method for large-scale microbiome analysis.

Physical Modeling of a Sliding Clamp Mechanism for the Spreading of ParB at Short Genomic Distance from Bacterial Centromere Sites.

SummaryBacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation are cytosine triphosphate (CTP)-dependent molecular switches. CTP-binding converts ParB dimers to DNA clamps, allowing unidimensional diffusion along the DNA. This sliding property has been proposed to explain the ParB spreading over large distances from parS centromere sites where ParB is specifically loaded. We modeled such a “clamping and sliding” mechanism as a typical reaction-diffusion system, compared it to the F plasmid ParB DNA binding pattern, and found that it can account neither for the long range of ParB binding to DNA nor for the rapid assembly kinetics observed in vivo after parS duplication. Also, it predicts a strong effect on the F plasmid ParB binding pattern from the presence of a roadblock that is not observed in ChIP-sequencing (ChIP-seq). We conclude that although “clamping and sliding” can occur at short distances from parS, another mechanism must apply for ParB recruitment at larger genomic distances.

Impact of smoking on heavy metal contamination and DNA fragmentation.

Tobacco is smoked by different techniques through cigarette and shisha smoking. The prevalence of tobacco is considered one of the major threats to public health. This study aims to assess the effect of cigarette, shisha, and mixed (cigarette/shisha) smoking on heavy metal contamination in hair samples, hair loss, and DNA fragmentation, to correlate age, incidence of hair loss, and smoking duration with the amount of accumulated metals and the DNA fragmentation, and to correlate the level of heavy metal contamination with DNA fragmentation. This study was implemented in Saudi Arabia among sixty males divided into four groups (15/group): control and cigarette, shisha, and mixed smokers. Heavy metal contamination in hair samples and urinary DNA levels were assayed. All metal and urinary DNA levels were significantly elevated in cigarette, shisha, and mixed smokers compared to non-smokers. Hair loss was also higher among smokers especially among participants with high DNA concentrations. There were positive significant correlations of age and incidence of hair loss with urinary DNA concentration. There were positive significant correlations between urinary DNA concentration and all heavy metal levels. Cigarette, shisha, and mixed smoking trigger metal contamination, DNA fragmentation, and hair loss. Moreover, hair loss was observed to be associated with Sb, Cd, and Ni as well as urinary DNA level, while age was associated only with lead and urinary DNA levels. The duration of smoking had a major impact on Pb and Sb levels. Finally, contamination with all six metals was significantly associated with DNA fragmentation.

High stream flows dilute environmental DNA (eDNA) concentrations and reduce detectability

AbstractAimEnvironmental DNA (eDNA) is a rapidly emerging methodology with important applications to environmental management and conservation. However, the effects of stream flow or discharge on eDNA have been minimally investigated in lotic (stream and river) environments. In this study, we examined the role of stream flow on eDNA concentrations and detectability of an invasive clam (Corbicula fluminea), while also accounting for other abiotic and biotic variables.LocationIllinois, United States of America.MethodsWe used a longitudinal study over a year in two streams, as well as a seasonal study (summer, autumn) in eight streams, to investigate the effects of variable stream flow on eDNA concentrations and detectability. We used linear mixed‐effects models to assess the influence of various factors on eDNA concentration and occupancy models to make predictions on how seasonality can influence eDNA detection.ResultsWe found higher stream flows decreased eDNA concentrations, and floods produced false negatives or non‐detections at locations where C. fluminea was relatively common. In addition, we found concentrations and detectability of C. fluminea eDNA to be higher in summer than in autumn.Main conclusionsWe found that stream flow dilutes eDNA concentrations, which may have serious implications for the detection of low abundance organisms. Managers and practitioners applying eDNA for rare species should seek to sample at low or base stream flows when feasible, and future studies should investigate whether our findings here are consistent for other taxa and lotic ecosystems.

High throughput DNA concentration determination system based on fluorescence technology

The measurement of DNA concentration is an important step in many research procedures in molecular biology. Traditional method for DNA concentration determination is based on ultraviolet absorbance which suffers from low sensitivity. Thus, we proposed a high throughput and low-cost DNA concentration system based on fluorescence technology. We analyzed the self-luminescence of each individual solution in DNA test kit, such as fluorescence dyes and DNA. It showed that the fluorescence emission from the dsDNA/dye complex was significantly higher than the self-luminescence from the single SYBR Green I, Gel Green and DNA solution when it was excited by laser diode source. SYBR Green I and Gel Green preferred CG bp sequences in dsDNA. dsDNA ranged from 2 to 15 ng/μL presented a stronger linear relationship with relative fluorescence intensity (R = 0.995). Such a low cost system could measure the concentration of 24 DNA samples within 100 s. It permits the estimation of concentrations of DNA as low as 2 ng/μL and offers a simple way for DNA concentration determination during biological research, especially for the labs in the third world countries.

Optimization and application of a high-resolution melting protocol in the characterization of avian infectious laryngotracheitis virus

A previous sequence analysis of a US5 gene fragment of infectious laryngotracheitis virus (ILTV) performed in an Argentinian epidemiological study allowed to differentiate between wild and vaccine strains. This analysis also defined five ILTV haplotypes with specific variations at positions 461, 484, 832, 878 and 894 of the US5 gene. This characterization of viral strains may also be accomplished using the High-Resolution Melting Analysis (HRMA), which has been described as an effective, fast and sensitive method to detect mutations in PCR products. In the present study, an HRM protocol was developed with the aim of characterizing the circulating ILTV strains in Argentina. The specificity of this tool was confirmed in different DNA diluents, without interference from heterologous DNA or other cellular metabolites. Additionally, the salt concentration in the elution buffer used for DNA extraction did not alter the curve profiles. Higher concentrations of DNA (Ct≅26.0) displayed well-defined curve profiles, whereas lower concentrations (Ct≅32.5) exhibited more heterogeneous curves. The HRMA showed 97.49% concordance with the reference technique, i.e., sequencing. The HRM protocol has the capability to perform DNA amplification prior to its characterization. Thus, eventually this technique may be used simultaneously as a diagnostic tool. This advantage implies a significant reduction in the time and effort involved in sample processing.

CtDNA Concentration, MIKI67 Mutations and Hyper-Progressive Disease Related Gene Mutations Are Prognostic Markers for Camrelizumab and Apatinib Combined Multiline Treatment in Advanced NSCLC.

Immunotherapy by immune checkpoint inhibitors (ICIs) has showed outstanding efficacy in the treatment of advanced non-small cell lung cancer (NSCLC). The combination of immunotherapy with anti-angiogenic therapy exhibited enhanced efficacy in multiline treatment. However, the potential biomarkers for predicting and monitoring the therapeutic response of the combined therapy remain undefined. In this study, we performed a pilot study by prospectively recruiting 22 advanced NSCLC patients who failed to previous lines of chemotherapy, chemoradiotherapy, TKI therapy, surgery, or any combination of the therapies, and investigated the prognostic factors for patients who received anti-PD-1 (Camrelizumab) and anti-angiogenic (Apatinib) combined therapy. The objective response rate (ORR) assessed by an independent radiology review was 22.7%, and the median progression-free survival (PFS) was 5.25 months. We found that high concentration of circulating-free DNA (cfDNA) (HR = 27.75, P = 0.003), MIKI67 mutation (HR = 114.11, P = 0.009) and gene variations related to hyper-progressive disease (HPD) (HR = 36.85, P = 0.004) were independent risk factors and exhibited significant correlation with PFS. Circulating tumor DNA (ctDNA) mutational status was also a predicting indicator for PFS. In contrast, the blood tumor mutational burden (bTMB) could not stratify the clinical benefit in this combined therapy (HR = 0.81, P = 0.137). Furthermore, we found that the variant allele fraction (VAF) of mutations in ctDNA was sensitive indicators of therapeutic response and therefore can be used to monitor the tumor relief or progression. In conclusion, cfDNA concentration, MIKI67 mutations and HPD-related mutations were independent risk factors and PFS predictors for multiline combined anti-angiogenic/ICI combined therapy. ctDNA may be a novel monitoring biomarker for therapeutic response and predicting biomarker for prognosis in future combined therapy involving PD-1 blockade.

Sus sp. DNA Encoding cyt b Gene Detection Test on Meat Grinding Samples Using Conventional PCR

Micro-entrepreneurs with basic ingredients of processed meat such as meatball who do not have a meat grinder, generally using meat grinder at the public market. The problem that occurs is that there is no clear regulation from the Government regarding the guarantee of the halal meat grinding in the Regional Company. This needs to be enhanced as a study, considering that the grinding material does not only come from Halal substances. The purpose of this study was to test pig DNA in meat grinding samples at PD Pasar Surya Surabaya City by using the conventional Polymerase Chain Reaction (PCR) method. DNA was isolated from 11 PD Pasar Surya meat grinding samples, then spectrophotometry was performed. Spectrophotometry results showed that all samples have high DNA concentrations. The primer used is the cyt b pig gene encoder. Predenaturation is performed at a temperature of 95°C-5 minutes, denaturation of 95°C-45 seconds, annealing 60°C-30 seconds, extension 72°C-40 seconds, and post extension 72°C-5 minutes. The results of PCR analysis were determined by the emergence of DNA bands of ± 149 bp as markers of pig DNA. The results showed negative on sample or no pig contamination in 11 samples tested. While the pig sample as positive control showed a band of ± 149 bp. These results prove that at 11 points of the location of meat grinding there is no contamination of pig DNA.