Specific Fragments Research Articles

Simultaneous detection of four specific DNAs fragments based on two-dimensional bimetallic MOF nanosheets

We have developed quantitative analysis method for simultaneous detection of four specific DNAs fragments based on two-dimensional bimetallic MOF nanosheets Co/Fe(1:1) MOF. In this analytical method, four different dyes were labeled on the complementary sequences of four different target DNAs (T-DNAs) as fluorescent probes, and Co/Fe(1:1) MOF was chosen as quencher. In the absence of T-DNAs, four fluorescent probes were adsorbed on the Co/Fe(1:1) MOF by π-π stacking, and the dyes were close to Co/Fe(1:1) MOF, their fluorescence is quenched by Co/Fe(1:1) MOF through photoinduced electron transfer. In the presence of T-DNAs, four different T-DNAs react with the corresponding fluorescent probe to form double strands DNA, which cannot be adsorbed by Co/Fe(1:1) MOF, and the fluorescence of dyes was recovered. Under the optimal conditions, the fluorescence intensities of four dyes all exhibited good linear dependence on corresponding concentration of T-DNAs. The detection limits of HAV, HBV, HCV and HIV were 0.032 nmol/L, 0.037 nmol/L, 0.53 nmol/L and 0.18 nmol/L (3σ, n = 9), respectively. The analytical method has low detection limit, good selectivity and high sensitivity, and can be used to detect T-DNAs in vitro.

Analysis of the bacterial flora of periodontal pockets and evaluation of the diagnostic value and the validity of using the PET test in the detection of Sockransky’s red complex pathogens at various stages and degrees of periodontitis

Introduction: In microbiological diagnostics, a significant role is played by the PET test based on a polymerase chain reaction developed thanks to medical engineering methods, which, by amplifying specific fragments of genetic material, enables the identification of microorganisms. As a consequence, it is possible to quantify and qualify a sample of biological material under study, which has found application in the diagnosis and treatment of periodontitis.
 Aim:The following paper shows the validity of carrying out the PET test in people with clinically diagnosed chronic periodontitis who also suffer from other loads, such as smoking or systemic diseases, as well as the physiological state of pregnancy.Another goal is also to confirm the presence of Sockransky's red complex bacteria responsible for the development of periodontitis.Material and Methods:The research was carried out at the Clinic of Conservative Dentistry and Periodontal Diseases, performing an examination of 4 patients, and then after determining the deepest periodontal pockets, biological material was collected from them and sent to a bacteriological laboratory, where it was analyzed.Results:The quantitative and qualitative determination of the samples of biological material collected from the pockets of the patients showed the presence of the red complex bacteria according to Sockransky, especially the species: Treponema denticola and Porphyromonas gingivalis.Conclusions: In the studied clinical cases, the PET test confirmed the presence of bacteria responsible for periodontitis. The legitimacy of its use was also confirmed, emphasizing that it is a tool that can be used by a dentist in implementing therapeutic decisions in the treatment of periodontitis.

New insights into the pathogenesis and molecular understanding of cutaneous T-cell lymphomas

The pathogenesis of cutaneous T‑cell lymphomas (CTCL) is still an enigma. Therefore, extensive translational research efforts have been undertaken in recent years to gain further clinical and molecular insights. There is increasing evidence that the different clinical appearance of the CTCL subtypes derives from the assumption that they develop from different skin subpopulations of Tcells. Detection and quantification of the malignant T‑cell clones is crucial for the diagnosis and prognosis of CTCL. Numerous recurrent mutant cellular signalling pathways have been found in recent years. This includes the JAK-STAT, NFκB, T‑cell receptor and MAP kinase signalling pathways, as well as cell cycle control and epigenetics. The most recent analyses imply atumour evolution model with initial copy number variation, like amplification or deletions of specific DNA fragments (CNVs) and only subsequent later single nucleotide variations (SNVs). The crucial question, however, is which CNVs are sufficient to initiate general tumourigenesis? The challenge is to identify possible driver genes. Increasing molecular understanding in CTCL will include new breakthrough therapeutic options in the near future.

Development of an MSPQC Nucleic Acid Sensor Based on CRISPR/Cas9 for the Detection of Mycobacterium tuberculosis.

Accurate and rapid detection of nucleic acid plays a vital role in the clinical treatment of tuberculosis caused by Mycobacterium tuberculosis (M.TB). However, false-negative and false-positive results caused by base mismatches could affect the detection accuracy. Inspired by the unique property of CRISPR/Cas9, we proposed a new MSPQC M.TB sensor based on the CRISPR/Cas9 system, which can distinguish single-base mismatches in 10 bases from the protospacer adjacent motif (PAM) region. In the proposed sensor, single-stranded DNA on Au interdigital electrodes was used as a capture probe for the target and an initiator for hybridization chain reaction (HCR). HCR was used to generate long double-stranded DNA (dsDNA), which could span the Au interdigital electrodes. CRISPR/Cas9 was used as recognition components to recognize capture/target dsDNA. When the target existed, the capture probe hybridized with the target to form dsDNA, which could be recognized and cut by CRISPR/Cas9. Thus, the DNA connection between electrodes was cut off and resulted in the MSPQC response. When no target existed, the capture probe remained single-stranded and could not be recognized and cut by CRISPR/Cas9. Therefore, DNA connection between electrodes was reserved. Moreover, silver staining technology was utilized to improve the sensitivity of detection. M.TB was detected by the proposed sensor using specific sequence fragments of 16S rRNA of M.TB as the target. The detection time was down to 2.3 h. The limit of detection (LOD) was 30 CFU/mL.

Prevalence of Cryptosporidium infection in cattle from selected commercial farms and nomadic settlements in Yola, Adamawa State

Cryptosporidium species are apicomplexan parasites commonly associated with diarrhoea in both men and animals. They are of public health importance. The study aimed to determine the occurrence of Cryptosporidium species in cattle in the Yola metropolitan area, Adamawa State, Nigeria. Four hundred and sixteen (416) faecal samples were collected from cattle in commercial farms and nomadic settlements and were analyzed using Modified Ziehl Neelsen (MZN) technique. Twenty-seven (27) positive samples were subjected to nested Polymerase Chain Reaction (PCR) for the amplification of a specific fragment of 18S rRNA gene that was used to detect Cryptosporidium spp. Seventy-three (17.5%) out of 416 samples were positive for Cryptosporidium oocysts by MZN, and 26 (96.0%) out of 27 isolated oocysts of Cryptosporidium detected by MZN were positive by PCR. Commercial farms had a higher prevalence (19.2%) of Cryptosporidium oocysts than the nomadic settlements (15.9%). Based on risk factors, there was a significant association (P<0.05) between the occurrence of Cryptosporidium oocysts and factors such as sex and faecal consistency. At the same time, age, breed, management system, animal source and drinking water source varied insignificantly (P>0.05). This study has shown the occurrence of Cryptosporidium infection in cattle in the Yola metropolis. There should be an awareness campaign among public health workers on the dangers posed by the organism to humans so that the Government would enforce control and preventive measures.

Fragment Localized Molecular Orbitals

We introduce the concept of fragment localized molecular orbitals (FLMOs), which are Hartree–Fock molecular orbitals localized in specific fragments constituting a molecular system. In physical terms, we minimize the local electronic energies of the different fragments, at the cost of maximizing the repulsion between them. To showcase the approach, we rationalize the main interactions occurring in large biological systems in terms of interactions between the fragments of the system. In particular, we study an anticancer drug intercalated within DNA and retinal in anabaena sensory rhodopsin as prototypes of molecular systems embedded in biological matrixes. Finally, the FLMOs are exploited to rationalize the formation of two oligomers, prototypes of amyloid diseases, such as Parkinson and Alzheimer.

Genetic transformation of LoHDZ2 and analysis of its function to enhance stress resistance in Larixolgensis

To study the function of LoHDZ2 in larch, we first constructed a VB191103-LoHDZ2::GUS overexpression vector. Through Agrobacterium-mediated infection, the expression vector was transferred into a larch embryogenic cell line. A stable resistant cell line was subsequently screened, and mature embryos were induced to grow until they developed into seedlings. Antagonistic cell lines were identified at both the DNA and RNA levels. The transgenic cell lines were then subjected to GUS staining, and transgenic cell lines were ultimately identified and obtained. These transgenic cell lines were sequenced to identify differentially expressed genes, and a cluster analysis was performed. The resistant cell lines were cultured under stress conditions involving 20% PEG6000 and 200 mM NaCl proliferation media (1/10-BM). After the stress treatment, the contents of peroxidase (POD), malondialdehyde (MDA) and superoxide dismutase (SOD) in both wild-type and transgenic cell lines were measured. The results are summarized below: (1) When the specific fragment of the target gene in the genome of the resistant cell line was amplified. At the RNA level, the expression of the fragment in four resistant lines increased. In addition, GUS staining showed a blue reaction, indicating that LoHDZ2 was successfully integrated into the larch embryonic cell lines. (2) To verify the accuracy and reliability of the transcriptome data, 10 differentially expressed genes (5 upregulated and 5 down regulated genes) were subjected to qRT-PCR verification. The results showed that the expression trend of the 10 differentially expressed genes was the same as that revealed by RNA-Seq, indicating that the transcriptome data were reliable. (3) The transcriptome sequencing showed that 176 genes were upregulated and that 140 genes were down regulated. Through GO enrichment analysis and KEGG metabolic pathway analysis, the screened differentially expressed genes were related to biological processes such as larch metabolism and response to stimuli, indicating that these genes may be closely involved in the regulation of the larch response to external stimuli, including heat stress, drought stress, metal ion stress and bacterial infection, and may participate in the growth process. (4) After 20% PEG6000 treatment, the POD enzyme activity of the transgenic cell line was greater than that of the wild-type; this activity could effectively remove the amount of peroxide produced. The MDA content of the transgenic cell lines was lower than that of the wild-type cell lines, and the accumulation degree of harmful substances was low, indicating that the degree of oxidative damage of the transgenic cell lines was lower than that of the wild-type cell lines. The SOD content of the transgenic cell lines was lower than that of the wild-type cell lines, indicating that the drought resistance of the transgenic cell lines was enhanced. After 200 mM NaCl treatment, although the increase in SOD content was not obvious, the same trend was detected, indicating that the resistance of the transgenic cell lines was indeed stronger than that of the wild-type cell lines. According to the results of previous experiments, after this gene was overexpressed in tobacco, the transformed plants showed obvious dwarfing, which may indicate that the stress resistance of the plant was enhanced. In conclusion, a transgenic larch cell line was successfully obtained, and transgenic larch seedlings were successfully induced. LoHDZ2 may participate in the response of plants to the external environment, and may participate in the growth and development of Larixolgensis by affecting plant metabolic pathways.

Biglycan neo-epitope (BGN262), a novel biomarker for screening early changes in equine osteoarthritic subchondral bone

Native biglycan (BGN), which can undergo proteolytic cleavage in pathological conditions, is well known to be involved in bone formation and mineralization. This study aimed to delineate the specific cleavage fragment, a neo-epitope for BGN (BGN262), in synovial fluid (SF) from young racehorses in training, osteoarthritic (OA) joints with subchondral bone sclerosis (SCBS), and chip fracture joints. A custom-made inhibition ELISA was developed to quantify BGN262 in SF. Cohort 1: A longitudinal study comprising 10 racehorses undergoing long-term training. Cohort 2: A cross-sectional study comprising joints from horses (N=69) with different stages of OA and radiographically classified SCBS. Cohort 3: A cross-sectional study comprising horses (N=9) with chip fractures. Receiver operating characteristic (ROC) curve analysis was performed (healthy joints vs chip joints) to evaluate BGN262 robustness. Cohort 1: SF BGN262 levels from racehorses showed a statistical increase during the first 6 months of the training period. Cohort 2: BGN262 levels were significantly higher in the SF from severe SCBS joints. Cohort 3: SF BGN262 levels in chip fracture joints showed a significant increase compared to normal joints. The ROC analysis showed an AUC of 0.957 (95% C.I 0.868-1.046), indicating good separation between the groups. The data presented show that BGN262 levels increase in SF in correlation with the initiation of training, severity of SCBS, and presence of chip fractures. This suggests that BGN262 is a potential predictor and a novel biomarker for early changes in subchondral bone (SCB), aiming to prevent catastrophic injuries in racehorses.

Honeybee Iflaviruses Pack Specific tRNA Fragments from Host Cells in Their Virions.

The Picornavirales include viruses that infect vertebrates, insects, and plants. It was believed that they pack only their genomic mRNA in the particles; thus, we envisaged these viruses as excellent model systems for studies of mRNA modifications. We used LC–MS to analyze digested RNA isolated from particles of the sacbrood and deformed wing iflaviruses as well as of the echovirus 18 and rhinovirus 2 picornaviruses. Whereas in the picornavirus RNAs we detected only N 6‐methyladenosine and 2’‐O‐methylated nucleosides, the iflavirus RNAs contained a wide range of methylated nucleosides, such as 1‐methyladenosine (m1A) and 5‐methylcytidine (m5C). Mapping of m1A and m5C through RNA sequencing of the SBV and DWV RNAs revealed the presence of tRNA molecules. Both modifications were detected only in tRNA. Further analysis revealed that tRNAs are present in form of 3’ and 5’ fragments and they are packed selectively. Moreover, these tRNAs are typically packed by other viruses.

Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in TH 2 responses.

Immune responses to N-glycan structures from allergens and parasites are often associated with pronounced, high affinity IgE reactivities. Cross-reactive carbohydrate determinants (CCDs) are constituted by modified N-glycan core structures and represent the most frequently recognized epitopes in allergic immune responses. Although recently accepted as potentially allergenic epitopes, the biological and clinical relevance as well as structural and functional characteristics of CCD-specific antibodies remain elusive. In order to gain structural insights into the recognition of CCDs, two specific antibody fragments were isolated from a leporid immune repertoire library and converted into human/leporid IgE and IgG formats. The antibody formats were assessed by ELISA and surface plasmon resonance, structural and functional analyses were performed by X-ray crystallography, mediator release, and ELIFAB assays. The recombinant IgE exhibited highly specific interactions with different types of CCDs on numerous CCD-carrying glycoproteins. Crystal structures of two CCD-specific antibodies, one of which in complex with a CCD-derived disaccharide emphasize that mechanisms of core glycan epitope recognition are as specific as those governing protein epitope recognition. The rIgE triggered immediate cellular responses via FcεRI cross-linking and mediated facilitated antigen presentation by binding of IgE/antigen complexes to CD23, a process that also could be blocked by IgG of allergic patients. Our study provides evidence for the relevance of N-glycan recognition in TH 2 responses and corroborates that IgE and IgG antibodies to ubiquitous carbohydrate epitopes can be equivalent to those directed against proteinaceous epitopes with implications for diagnostic and immunotherapeutic concepts.

DEVELOPMENT OF SPECIFIC PRIMERS FOR 16S rRNA GENE ANALYSIS IN THE DETECTION OF Ralstonia solanacearum SPECIES COMPLEX

Members of Ralstonia solanacearum species complex (RSSC) are causal agents of vascular wilt disease in more than 450 crop species, including solanaceous plants such as potatoes, tomatoes, bell pepper, eggplant, etc. These phytopathogens cause serious yield loss mostly in solanaceous crops which are grown in tropical, subtropical, and temperate regions of the world. Yield losses comprise 80%–100% in potato, up to 91% for tomato, 10%–30% in tobacco, 33%–90% in banana, and reduce crop productivity and yield. PCR-methods are specific, sensitive and cost-effective approaches for the detection and identification of RSSC members. The objective of this study was to compare specificity of routinely used primer mix for PCR RSSC detection with the newly developed pairs of species-specific primers for ease of use diagnostics in a laboratory. Materials and Methods. The conserved genomic regions of the 16S rRNA sequences of R. solanacearum, R. pseudosolanacearum, and R. syzygii were selected for the design of primers for this study. Newly created primer species specificity was tested in PCR using the DNA of the two targets and 13 non-target strains of bacteria. Results. Three pairs of newly created primers Rs-28(F)/Rs-193(R), Rs-28(F)/OLI-160(R), Rs28(F)/OLI248(R) produced single specific fragments for bacterial strains of Ralstonia solanacearum: 166 bp, 132 bp, and 220 bp. products respectively. No PCR products were obtained during amplification with the negative control or non-target DNA templates from other bacterial species. Conclusion. Designed primers can be used for the development of PCR system for the qualitative and quantitative detection of RSSC members.

Psychological Emotion and Behavior Analysis in Music Teaching Based on the Attention, Relevance, Confidence, and Satisfaction Motivation Model.

The abbreviation ARCS in the ARCS motivational model comprises the first letters of four English words: Attention, Relevance, Confidence, and Satisfaction. The ARCS motivation model is based on a systematic and easy-to-operate motivation theory. Many research studies have verified the applicability and effectiveness of the ARCS model in education worldwide. The proposed optimized ARCS motivation model takes the traditional ARCS motivation model and systematically optimizes it to make it suitable for the coding of data from videos of music classes. The case analysis method selected 14 high-quality music lessons as the research object. The optimized ARCS model involves text conversion, coding, and statistical analysis of the transcripts of videos of high-quality music classes. The data analysis was used to analyze the characteristics of students’ emotional and behavioral motivation incentive strategies in each section of the ARCS motivation model in combination with specific high-quality case lesson fragments. This paper summarizes ways and means of using a motivational approach in the teaching of quality music classes, providing theoretical support for the practice of music teaching.

PB2042: CLINICAL AND MOLECULAR CORRELATION OF PATIENTS WITH ESSENTIAL THROMBOCYTHEMIA: A RETROSPECTIVE ANALYSIS FROM A TERTIARY CARE CENTER IN INDIA.

Background: Approximately 50–70% of patients of ET harbor JAK2 mutation while about 20–30% patients carry CALR mutations. Data from developed countries has shown that CALR mutated patients have higher platelet count and lower thrombotic risk compared to JAK2 mutated patients. In India, testing for CALR and MPL mutation and next generation sequencing has become available only in last 5 years. There is lack of data regarding mutation status and its clinical correlation. We studied clinical features of ET patients presented to our hospital according to their mutation status. Aims: To study the molecular mutations and correlate them with the clinical features in ET patients Methods: We did the retrospective analysis of the patients diagnosed as ET from January 2016 to December 2021. Allele Specific PCR and fragment analysis was done to detect JAK2 (V617F), MPL (W515L/K) and CALR exon-9 mutations. Risk stratification done as per revised IPSET (International Prognostic Scoring for thrombosis in Essential Thrombocythemia) score. Therapy was started in all the patients according to their risk score with hydroxyurea or aspirin as the standard of care. Results: A total of 39 diagnosed cases of ET were identified. Median age was 55 years (30-83), 20/39 males (51.3%) and 19/39 females (48.7%). JAK2 mutation was found in 25.6% (10/39), CALR in 38.5% (15/39) and MPL in 5.1% (2/39) cases. Triple negativity was found in 30.8% (12/39) cases. Very low, low, intermediate and high-risk patients as per IPSET score were 53.9% (21/39), 10.2% (4/39), 20.5% (8/39) and 15.4% (6/39) respectively. The median age was statistically significantly lower in CALR positive cases compared to the JAK2 positive cases (45.9 vs 60.9years, p=0.011). The median Hemoglobin and platelet counts were slightly higher in CALR positive group (12.3 g/dL and 10.15x109/l) compared to JAK2 mutated and triple negative groups but the difference was not statistically significant. Three patients (7.7%) presented with thrombosis; two with cerebral venous thrombosis both CALR positive, one with arterial stroke and MPL gene mutation. Ten (25.6%) patients were lost to follow up. One (2.6%) died to pulmonary infection. Median follow up period was 24 months. None of the patient transformed to acute myeloid leukemia or myelofibrosis. None of the patient developed thrombotic event after diagnosis and appropriate treatment. Summary/Conclusion: We observed that CALR was the most common mutation in our study. The CALR mutated patients were younger in age but the median platelet counts, WBC count and Hemoglobin were not statistically different among different mutation groups. In contrast to western studies, thrombosis was observed to be higher in CALR mutated group than others in our patient population. We recommend larger multicenter study for further elucidation of these observations.

Evaluation of the g473a polymorphism in the lysyl oxidase gene as a risk factor related to the occurrence of breast cancer in brazilian women

Introduction: breast cancer is the most diagnosed type of cancer and the leading cause of death among women worldwide. Approximately 1.67 million new cases of breast cancer were diagnosed in 2012, leading to more than half a million deaths. Breast cancer accounted for 11.6% of newly diagnosed cancers (2,089 million) and 9.2% (787,000) of cancer-related deaths for both sexes and at all ages worldwide in 2018. Objective: breast cancer, as the most diagnosed carcinoma in the world and the leading cause of death among women, is a morbidity of outstanding importance, and the object of this study is to evaluate the association between the LOX gene G473A (rs1800449) polymorphism and breast cancer occurrence, potentially establishing a new finding in the identification of risks, prevention, and care for a specific group of women. Methods:in this retrospective cohort study, LOX G473A polymorphism frequency was assessed in 148 women with breast cancer and 245 women without breast cancer. All patients completed a questionnaire to identify possible risk factors and subsequently underwent peripheral blood collection to study the LOX gene. DNA was extracted followed by gene amplification via PCR, and the polymorphism was studied by specific fragment electrophoresis after digestion of the samples with the restriction endonuclease Pstl. Results: this study identified the use of oral contraceptives and family history of breast cancer as risk factors for breast cancer; the G473A polymorphism in LOX was not identified as a risk factor. Conclusion: a relationship was not observed between the LOX G473A polymorphism and the occurrence of breast cancer.

The Genetic Diversity of Bletilla spp. Based on SLAF-seq and Oligo-FISH.

Bletilla spp. Rchb. F. is a traditional Chinese medicinal material. In this study, Bletilla striata (Thunb. ex A. Murray) Rchb F, Bletilla formosana (Hayata) Schltr, and Bletilla ochracea Schltr were collected to analyze the genetic diversity of 16 materials using specific site-amplified fragment sequencing (SLAF-seq) and fluorescence in situ hybridization (FISH). The results showed that the phylogenetic tree of the single-nucleotide polymorphism (SNP) data rendering system was correlated with the shape and geographical distribution of the material. The results of the population structural analysis showed that all the materials containing yellow labellum came from the same ancestor. The results of the principal component analysis were able to preliminarily judge the genetic distance and provided a reference for the selection of hybrid parents. The FISH analysis showed that the chromosomes of B. striata were 2n = 32 and the chromosomes of the B. striata (safflower) mutant were 2n = 34 and the chromosomes of B. ochracea and B. formosana were 2n = 34–36. The (AG3T3)3 non-terminal signal was different from the 5S rDNA signal. These results revealed that the 16 materials had rich genetic diversity, which can provide molecular and cytogenetic data for the study of the genus and its relatives and serve as a reference for the breeding of new genus varieties and improve breeding efficiency and cost.

Genome-Wide Association Analysis and Gene Mining of Resistance to China Race 1 of Frogeye Leaf Spot in Soybean.

Soybean frogeye leaf spot (FLS) is a worldwide fungal disease. Its higher occurrence frequency and wider distribution range always led to severe yield losses of soybean, therefore, breeding new cultivars with FLS resistance has been an important breeding goal for soybean breeders. In this study, an association panel of 183 representative soybean accessions was used to evaluate their resistance to FLS race 1, and to identify quantitative trait nucleotides (QTNs) and candidate genes based on genome-wide association study (GWAS) and high-throughput single-nucleotide polymorphisms (SNPs). A total of 23,156 high-quality SNPs were developed using the specific locus-amplified fragment sequencing (SLAF-seq) approach. Finally, 13 novel association signals associated with FLS race 1 resistance were identified by the compressed mixed linear model (CMLM). In addition, 119 candidate genes were found within the 200-kb flanking genomic region of these 13 peak SNPs. Based on the gene-based association analysis, haplotype analysis, expression pattern analysis, and virus-induced gene silencing (VIGS) systems, four genes (Glyma.05G121100, Glyma.17G228300, Glyma.19G006900, and Glyma.19G008700) were preliminarily proved to play an important role in the soybean resistance to FLS race 1.

A Novel PD-L1-Containing MSLN Targeting Vaccine for Lung Cancer Immunotherapy.

Therapeutic tumor vaccines have become an important breakthrough in the treatment of various solid tumors including lung cancer. Dendritic cells (DCs)-based tumor vaccines targeting tumor-associated antigens (TAAs) play a key role in immunotherapy and immunoprevention. However, the weak immunogenicity of TAAs and low immune response rates are a major challenge faced in the application of therapeutic tumor vaccines. Here, we tested whether targeting an attractive target Mesothelin (MSLN) and PD-L1 immune checkpoint molecule to DCs in vivo would elicit therapeutic antitumor cytotoxic T lymphocyte (CTL) response. We generated specific MSLN fragment combined with PD-L1 and GM-CSF peptide immunogen (MSLN-PDL1-GMCSF) based on the novel anti-PD-L1 vaccination strategy we recently developed for the cancer treatment and prevention. We found that DCs loaded with MSLN-PDL1-GMCSF vaccine elicited much stronger endogenous anti-PD-L1 antibody and T cell responses in immunized mice and that antigen specific CTLs had cytolytic activities against tumor cells expressing both MSLN and PD-L1. We demonstrated that vaccination with MSLN-PDL1-GMCSF potently inhibited the tumor growth of MSLN+ and PD-L1+ lung cancer cells, exhibiting a significant therapeutic anti-tumor potential. Furthermore, PD-1 blockade further improved the synergistic antitumor therapeutic efficacy of MSLN-PDL1-GMCSF vaccine in immunized mice. In summary, our data demonstrated for the first time that this PD-L1-containing MSLN therapeutic vaccine can induce persistent anti-PD-L1 antibody and CTL responses, providing an effective immunotherapeutic strategy for lung cancer immunotherapy by combining MSLN-PDL1-GMCSF vaccine and PD-1 blockade.

Abstract 13: Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro

Abstract Despite the well-documented benefits of circulating tumor DNA (ctDNA) based liquid biopsy, including screening, prognostication, and treatment monitoring, there remain several concerns with its application. Chief among these concerns are imperfect sensitivity and specificity, particularly pre-diagnosis and after tumor resection, when ctDNA burden is low. Cell death, either through apoptosis or necrosis, has been the most widely accepted mechanism of DNA release. Recent studies have also suggested active release through extracellular vesicles as a paradigm for DNA release. However, this pathway remains highly debated in terms of its contribution, mechanism, and even credibility. To address the deficiencies in ctDNA detection in the clinic and the lack of basic mechanistic knowledge of cf/ctDNA release, we performed an in vitro CRISPR screen on MCF10A cells to identify regulators of DNA release. This normal breast epithelial cell line was chosen because MCF10A cells release DNA into cell culture media with a similar fragmentation pattern to that observed in human blood. After lentiviral infection with the genome-wide Brunello CRISPR-Cas9 knockout library, the population of knockouts identified in the genomic DNA of cells attached to the plates was compared to the population of knockouts found in the DNA released into the cell-depleted culture media. Genes with skewed representation were considered potential regulators of DNA release. Our analysis identified several genes in the TRAIL apoptotic pathway including FADD, as well as several genes encoding RNA-binding proteins including Sam68. Knockout of Sam68 and FADD in MCF10As, MDA-MB-468 breast cancer cells, and NCI-H841 lung cancer cells lead to decreases in DNA release. Previous studies have hypothesized that the fragment size of released DNA is dependent on release mechanism. However, the decreases observed in our knockout cell lines were not limited to a specific fragment size of DNA. Application of the small molecule apoptosis inhibitor Z-VAD-FMK also recapitulated the DNA release phenotype seen in our knockout cell lines. From these results, we conclude that apoptosis is the major source of DNA release from cancer cells, and that fragment size is not the indicator of release mechanism it has been presumed to be. Mechanistic studies of DNA release such as this could lead to the ability to modify the amount of ctDNA in circulation, which would significantly improve accurate detection in the clinic. Citation Format: Brad A. Davidson, Adam X. Miranda, Sarah Croessmann, Ben Ho Park. Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 13.

A few words about dignity: An introspective study of a person with disability

In the article, I present issues of presence, existential absence, and the sense of dignity of a person with a disability. I present these issues for the first time and attempt to show disability “from within”, a perspective to which I am no stranger as I myself am disabled. The issues of presence, absence and human dignity have always fascinated me. Hence, in this text, they are “filtered” through my dramatic and sometimes even traumatic personal experiences. In conveying these experiences, I never forget what human existence should be – the fact that there should be dignity, presence and being. Is my human experience marked by this? At some point in the text I make a “moral accusation” in connection with this issue. Additionally, I have never forgotten (and will never forget) the person who helped me to “reborn” for myself, people and the world. “The significant person and her achievement in relation to the author” is my modest expression of gratitude to someone because of whom I LIVE. However, the central points of reference in this text are questions of human dignity and presence. They form its axis and are its guiding categories of analysis. I conclude by stating that these are important, even essential issues in order to understand the phenomenon of what it is to be human. And to be unique in that humanity. The main research question which I pose is the following: Does society see that people with disabilities have dignity? Furthermore, the thesis I propose and which I believe arises from this question is: society has little recognition of the dignity of people with disabilities. More specific research questions will be quoted below in the text which, in terms of methodology, has been written using an autoethnographic approach (Wolcott 2004, Kacperczyk 2014, Urbańska 2012). The choice of this highly qualitative methodology was guided by the assumption that autoethnography allows a subjective presentation of specific fragments of those events inscribed in the category of human dignity described here. This will be an analytical autoethnography, after Canagarajah (2012), because through writing I analyze the emotional, mental and situational states that I have had to face in the past, while some of the experiences that generated these states resonate with me to the present. Last but not least, it is my hope that my openly described states of mind will contribute to some betterment of this particular area of human existence in its extremely important, social and cultural dimensions.

Generation of Matrix Degradation Products Using an In Vitro MMP Cleavage Assay.

Matrix metalloproteinases (MMPs) play crucial roles in tissue homeostasis and pathologies by remodeling the extracellular matrix. Previous studies have demonstrated the biological activities of MMP-derived cleavage products. Furthermore, specific fragments can serve as biomarkers. Therefore, an in vitro cleavage assay to identify substrates and characterize cleavage patterns could provide important insight in disease-relevant mechanisms and the identification of novel biomarkers. In the pathogenesis of osteoarthritis (OA), MMP-2, -8, -9 and -13 are of vital importance. However, it is unclear which protease can cleave which matrix component. To address this question, we established an in vitro cleavage assay using recombinantly expressed MMPs and the two cartilage matrix components, COMP and thrombospondin-4. We found a time- and concentration-dependent degradation and an MMP-specific cleavage pattern for both proteins. Cleavage products can now be enriched and purified to investigate their biological activity. To verify the in vivo relevance, we compared the in vitro cleavage patterns with serum and synovial fluid from OA patients and could indeed detect fragments of similar size in the human samples. The cleavage assay can be adapted to other MMPs and substrates, making it a valuable tool for many research fields.