Tapestry: A Single-Round Smart Pooling Technique for COVID-19 Testing

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying.

A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain, the Omicron variant (Pango lineage B.1.1.529), was identified in South Africa in late September 2021. This variant has multiple spike protein deletions and mutations, with 15 amino acid substitutions detected in the receptor-binding domain (RBD). These RBD substitutions are hypothesized to increase infectivity and reduce antibody affinity, which is supported by recent data showing that the Omicron variant spreads faster than the Delta variant (Pango lineage B.1.617.2). Thus, this increase in infectivity should lead to Omicron being the dominant variant and developing screening tests that discriminate between Omicron and Delta variants is urgently needed. In this study, we successfully developed a novel screening assay using high-resolution melting analysis, in which two genotypes at G446/L452 and S477/T478 RBD were determined (G446S/L452 and S477N/T478K for Omicron; G446/L452R and S477/T478K for Delta). Using synthetic DNA fragments, we confirmed both melting point and melting peak shape of the RBD Omicron variant was distinguishable from those of wild-type and the Delta variant. Although this study was conducted without clinical samples, these results suggest that our high-resolution melting (HRM)-based genotyping method can readily identify the Omicron and Delta variants. This simple method should contribute to the rapid identification of SARS-CoV-2 variants and thus prevent potential widespread infection and inflow of the Omicron variant.

The COVID-19 pandemic has significantly affected adolescents' mental health, raising concerns about the potential increase in self-harming and suicidal behaviors (SIB). This study aimed to evaluate the pandemic's impact on SIB among adolescents and explore the effects of school closures and psychiatric vulnerabilities to inform future interventions. A systematic review and meta-analysis were conducted using PubMed, resulting in 420 studies, of which 36 studies (comprising 53 samples) met the inclusion criteria, encompassing 1,754,106 adolescents. Effect sizes were calculated using proportion effect size. Heterogeneity across studies was assessed using Q-statistics and I², while Egger's test was applied to evaluate publication bias. Differences between clinical and community samples, as well as the influence of psychiatric symptoms or disorders, were examined. Clinical samples demonstrated higher rates of SIB compared to community samples, with adolescents having psychiatric symptoms or disorders being more vulnerable. Overall, the pandemic was associated with a decline in SIB rates compared to pre-pandemic levels. Suicidal ideation decreased by 4.14% in combined samples and by 7.13% in community samples. Self-harm rates fell by 5.38% and 6.62% in combined and community samples, respectively. In contrast, clinical samples showed a distinct trend, with suicidal ideation decreasing by 29.39%, while self-harm rates increased by 32.51% and suicide attempts rose by 22.41%. The COVID-19 pandemic's impact on adolescents' SIB varied significantly across population types and levels of psychiatric vulnerability. While general declines were observed, the pronounced increases in self-harm and suicide attempts among clinical samples highlight the need for tailored mental health interventions. Addressing these disparities is essential for future mental health strategies and crisis preparedness.

From the outbreak of the novel coronavirus 2019 (COVID-19) a new physical and social distancing environment has changed our lives and, more particularly, the way of perceiving oneself, as well as eating attitudes and behaviors. An increasing number of studies have highlighted a risky scenario in terms of negative perceptions of one's body as well as disordered eating and eating disorder patterns in both clinical and general population. With regard to this postulate, this literature review posits two main concepts-perceptual disturbances and dysfunctional eating attitudes and behaviors-in the general and (sub-)clinical populations, to provide an understanding of these phenomena during the COVID-19 pandemic. The main objective of this article is to provide a comprehensive and critical review of published scientific literature about perceptual disturbances (i.e., negative body image, body image disturbances, low body esteem) and dysfunctional eating attitudes and behaviors, including disordered eating (e.g., restrictive eating, binge-eating episodes, overeating, emotional eating) and eating disorders features in community (i.e., general population) and clinical and sub-clinical samples worldwide during the COVID-19 pandemic. The PubMed, ScienceDirect, Ebsco, and Google Scholar databases were searched. The initial search produced 42 references. Scientific publications from March 2020 to April 2022 were included, and among the works compiled, only published research articles have been retained. Purely theoretical papers were also excluded. The final selection consisted of 21 studies, covering both community, clinical (i.e., eating disorder population), and sub-clinical samples. The details of the results are discussed taking into consideration the potential impact of changes in the way we perceive ourselves and interact with others (e.g., the popularity of videoconferencing and the over-use of social network sites due to social isolation) as well as changes in eating attitudes and behaviors, physical activity and exercise (e.g., as an emotional response to the insecurity generated by the pandemic context), in community and (sub-)clinical samples. The discussion sheds light on two outcomes: (1) a summary of findings with methodological considerations; (2) an intervention continuum to deal with the consequences of the COVID-19 pandemic; (3) and a final conclusion.

BackgroundIn Italy the pandemic of COVID-19 infection has placed an enormous burden on health authorities: contact precautions are required to avoid viral transmission and people should be subjected to standard infection control procedures. This is crucial in a country experiencing a high number of confirmed cases of COVID-19 infection in Europe and where multidrug-resistant Gram-negative bacteria (MDR-GN) are endemic.The aim of this study was to compare the prevalence of MDR-GN in surveillance rectal swabs (SRS) and in clinical samples (CS) in the period March 1,2020-April, 24 2020 with respect to the previous 2-month period and to the previous year.MethodsThe first SRS and the first CS with a MDR-GN isolate detected from 01/01/2019 to 24/04/2020 were included. Analysis was made by comparing three different study periods in 2019 and 2020 (Jan-Dec 2019, Jan-Feb 2020, and Mar-Apr 2020), for medical department, surgical department and intensive care department.ResultsOverall, 612 MDR-GN organisms were identified (399 SRS and 213 CS): carbapenemase-producing Klebsiella pneumoniae and Acinetobacter baumanii (CPAB) were the most frequently detected (Figure 1). We observed an increased relative frequency of patients with MDR-GN detected in CS respect to those found in SRS (32.7% vs 44.5% vs 70.6%, p=0.0005): 5/12 CS detected in the last period were isolated from the respiratory tract (Figure 2). Nine patients with COVID-19 pneumonia had MDR-GN. All but two patients had a previous negative SRS performed 4 days before (median value) and the median interval between COVID-19 positivity and MDR-GN positivity was 7 days.The six patients with CPAB isolation were all hospitalized in the same ward, with partially overlapping hospital stays during the study period. In 5 of them, CPAB was detected in the respiratory tract (Figure 3).ConclusionThe first detection of MDR-GN in CS and the nosocomial MDR-GN acquisition despite cohorting due to COVID-19 infection underline the need to reinforce infection control measures in a high prevalence country during COVID-19 pandemia. A correct antimicrobial policy urged because, according to published data, most patients with COVID-19 infection received antimicrobial therapy: furthermore MDR-GN infection could play a role in the negative outcome of these patients.DisclosuresAll Authors: No reported disclosures

A nanoparticle-based molecular beacon for directly detecting attomolar small RNA from plasma without purification

Quantitative RT-PCR tests that measure transcript abundance of selected genes in clinical specimens promise to improve cancer diagnostic accuracy and enable “personalized medicine” through selection of the most effective treatment for each cancer. However, most clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE) and these yield sub-optimal RNA integrity. Thus, there is a need for qPCR tests with increased robustness that also have intrinsic quality control and low cost. To address this need, we developed qPCR methods that enable simultaneous measurement of each target gene and reference gene transcript relative to a known number of gene-specific internal standard (IS) molecules using two-color fluorometric analysis on real-time platform. For each gene, a competitive template IS was synthesized containing 4-6 nucleotide changes relative to the native template (NT). During PCR, NT or IS signal was quantified with sequence-specific FAM-labeled probe or Quasar 670-labeled probe, respectively. To optimize measurement sensitivity for analysis of highly degraded FFPE samples; a) each RNA sample was reverse transcribed (RT) with gene-specific primer and b) cDNA was pre-amplified with 18 PCR cycles in the presence of internal standards. Results obtained with pre-amp or no pre-amp conditions were highly correspondent. We conducted a validation study of this assay in 38 samples (10 malignant and 10 benign surgical FFPE tissues and 13 malignant and 5 benign fine needle aspirate (FNA) samples. Consistent with previous results in fresh frozen surgical samples, the lung cancer diagnostic test (LCDT) optimal cut-off value discriminated malignant from non-malignant tissues (p = 0.0009), had 92.9% specificity and 75.0% sensitivity, and a receiver operator characteristic area under the curve of 0.87 (95% confidence interval 0.74-0.99). Based on these data, we expect that this quality-controlled two color fluorometric qPCR approach will enable reliable analysis of the LCDT and other promising qPCR-based molecular diagnostic tests in small degraded RNA extracted from clinical FFPE and FNA cell block FFPE samples. Citation Format: Jiyoun Yeo, Erin L. Crawford, Thomas M. Blomquist, Lauren M. Stanoszek, Rachel E. Dannemiller, Laura Jordan, Jill Zyrek, Luis de la Casas, James C. Willey. Use of two-color fluorometric real-time PCR to develop molecular diagnostic tests with intrinsic quality control that augment cytomorphologic diagnosis of lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 61. doi:10.1158/1538-7445.AM2013-61

It is reported on solid-phase chemical degradation sequencing of lore than 50 synthetic DNA fragments containing various 5-substituted pyrimidines including uracil (U), 5-fluorouracil (F5U), 5-bromouracil (Br5U), α and β anomers of 5-propyluracil (α-prop5U and B-prop5U) and 5-methylcytosine (a5C), Characteristic sequence patterns of oligonucleotides containing these base analogues are presented and their modification behaviour discussed

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology, and synthetic biology. We developed an error-correcting recursive construction procedure that attempts to address this challenge. Making DNA molecules from synthetic oligonucleotides using the procedure described here surpasses existing methods for de novo DNA synthesis in speed, precision, and amenability to automation. It provides for the first time a unified DNA construction platform for combining synthetic and natural DNA fragments, for constructing designer DNA libraries, and for making the faultless long synthetic DNA building blocks needed for de novo genome construction.

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

It's Diagnostics, Stupid

Background Monkeypox is a double-stranded DNA virus (approximately 197 kbp) belonging to the genus Orthopoxivirus. It is responsible for sporadic outbreaks in countries in Africa. Recently, in May 2022 a patient with a recent travel history to Nigeria tested positive for monkeypox in the UK. After that, people with symptoms (usually epithelial pustular lesions) were diagnosed with the virus in several countries, such as Portugal and the United States. In Brazil, the first case was confirmed in June 2022, the patient had traveled to Spain and Portugal. It was in this context that the Technical Operational Nucleus of our laboratory, Sabin Diagnosis and Health, located in Brasilia, capital of Brazil, validated and implemented a test to detect the genetic material of Monkeypox. Thus, in this work we present the implementation of this test. Method For the detection of Monkeypox viral DNA we chose to implement a real-time PCR (qPCR) methodology. The validated samples were swabs and wound scabs. The nucleic acid extraction chosen was using the Virus Pathogen kit from (QIAGEN), ‘midi’ protocol in the qiasymphony equipment (QIAGEN). Later we validated the extraction with the Maxwell RSC Viral Total Nucleic Acid Purification Kit extraction kit (Promega). For qPCR, we selected monkeypox-specific primers and probes, which were not able to recognize other Orthopoxiviruses (such as human pox) to ensure specificity. We used RNASEP-specific primers as an endogenous extraction control. As a positive control (PC) we used a synthetic double-stranded DNA fragment with the target sequence flanked by 10 bp. The limit of detection of the test was performed using serial dilutions of the positive control. Furthermore, given the initial absence of a positive sample for validation, we designed four pairs of primers that amplified four different regions of the viral genome for sequencing by the Sanger technique. The first positive samples were confirmed by sequencing these four regions. Results The protocol was designed in the first week after the confirmation of the first case in Brazil. The first tests with the PC showed the effectiveness of the primers. After adjusting the reaction conditions to assess the specificity of the test, we tested the protocol with samples known to be positive for other viral pathogens and obtained negative results for all of them, confirming the high specificity. After that we experimentally determined the limit of detection (25 copies per reaction). On July 8, 2022, one month after the first confirmed case in Brazil in the state of São Paulo, we released the Monkeypox detection test with the methodology described above. Since then, we have performed 193 tests, of which 59 (31%) were positive, most of them (56–95%) in men. The first 10 positive results were confirmed by genetic sequencing (Sanger). Discussion The implementation of the Monkeypox detection test by our laboratory allowed a quick response for Brazilian patients who needed the test after the virus entered the country.

BackgroundSexually transmitted infections (STIs) are prevalent throughout the world and impose a significant burden on individual health and public health systems. Missed diagnosis and late treatment of STIs can lead to serious complications such as infertility and cervical cancer. Although sexually transmitted co-infections are common, most commercial assays target one or a few STIs. The HPV-STI ChapterDx Next Generation Sequencing (NGS) assay detects and quantifies 29 HPVs and 14 other STIs in a single-tube and single-step PCR reaction and can be applied to tens to thousands of samples in a single sequencing run.MethodsA cohort of 274 samples, previously analyzed by conventional cytology/histology and Roche cobas HPV Test, were analyzed by ChapterDx HPV-STI NGS assay for detection of 43 HPV and STI. A set of 43 synthetic control DNA fragments for 43 HPV and STI were developed to evaluate the limit of detection, specificity, and sensitivity of ChapterDx HPV-STI NGS assay.ResultsThe assay was evaluated in this study, and the limit of detection was 100% at 50 copies for all targets, and 100%, 96%, 88% at 20 copies for 34, 8, and 1 target, respectively. The performance of this assay has been compared to Roche cobas HPV test, showing an overall agreement of 97.5% for hr-HPV, and 98.5% for both, HPV16 and HPV18. The assay also detected all HPV-infected CIN2/3 with 100% agreement with Roche cobas HPV results. Moreover, several co-infections with non-HPV STIs, such as C. trachomatis, T. vaginalis, M. genitalium, and HSV2 were identified.ConclusionsThe ChapterDx HPV-STI NGS assay is a user-friendly, easy to automate and cost-efficient assay, which provides accurate and comprehensive results for a wide spectrum of HPVs and STIs.

Two hybrid promoters that are functional in Escherichia coli have been constructed. These hybrid promoters, tacI and tacII, were derived from sequences of the trp and the lac UV5 promoters. In the first hybrid promoter (tacI), the DNA upstream of position -20 with respect to the transcriptional start site was derived from the trp promoter. The DNA downstream of position -20 was derived from the lac UV5 promoter. In the second hybrid promoter (tacII), the DNA upstream of position -11 at the Hpa I site within the Pribnow box was derived from the trp promoter. The DNA downstream of position -11 is a 46-base-pair synthetic DNA fragment that specifies part of the hybrid Pribnow box and the entire lac operator. It also specifies a Shine-Dalgarno sequence flanked by two unique restriction sites (portable Shine-Dalgarno sequence). The tacI and the tacII promoters respectively direct transcription approximately 11 and 7 times more efficiently than the derepressed parental lac UV5 promoter and approximately 3 and 2 times more efficiently than the trp promoter in the absence of the trp repressor. Both hybrid promoters can be repressed by the lac repressor and both can be derepressed with isopropyl beta-D-thiogalactoside. Consequently, these hybrid promoters are useful for the controlled expression of foreign genes at high levels in E. coli. In contrast to the trp and the lac UV5 promoters, the tacI promoter has not only a consensus -35 sequence but also a consensus Pribnow box sequence. This may explain the higher efficiency of this hybrid promoter with respect to either one of the parental promoters.

The enzyme 1-amino-cyclopropane-1-carboxylic acid synthase (ACS) participates in the ethylene biosynthesis pathways and it is tightly regulated transcriptionally and post-translationally. Notwithstanding its major role in climacteric fruit ripening, the transcriptional regulation of ACS during ripening is not fully understood. We studied fruit ripening in two Japanese plum cultivars, the climacteric Santa Rosa (SR) and its non-climacteric bud sport mutant, Sweet Miriam (SM). As the two cultivars show considerable difference in ACS expression, they provide a good system for the study of the transcriptional regulation of the gene. To investigate the differential transcriptional regulation of ACS1 genes in the SR and SM, their promoter regions, which showed only minor sequence differences, were isolated and used to identify the binding of transcription factors interacting with specific ACS1 cis-acting elements. Three transcription factors (TFs), abscisic acid-insensitive 5 (ABI5), GLABRA 2 (GL2), and TCP2, showed specific binding to the ACS1 promoter. Synthetic DNA fragments containing multiple cis-acting elements of these TFs fused to β-glucuronidase (GUS), showed the ABI5 binding site mediated ethylene and abscisic acid (ABA) responses of the promoter. While TCP2 and GL2 showed constant and similar expression levels in SM and SR fruit during ripening, ABI5 expression in SM fruits was lower than in SR fruits during advanced fruit ripening states. Overall, the work demonstrates the complex transcriptional regulation of ACS1.