Minus Strand Research Articles

Effect of Pregenomic RNA on the Mechanical Stability of HBV Capsid by Coarse-Grained Molecular Simulations.

Hepatitis B virus (HBV) is a double-stranded DNA virus, but its life cycle involves an intermediate stage, during which pregenomic RNA (pgRNA) is encapsulated in the capsid and then reverse-transcribed into the minus DNA strand. These immature HBV virions are the key target for antiviral drug discovery. In this study, we investigate the flexibility and mechanical stability of the HBV capsid containing pgRNA by employing residue-resolved coarse-grained molecular dynamics simulations. The results showed that the presence of pgRNA tends to decrease the overall flexibility of the capsid. In addition, the symmetrically arranged subunits of the capsid show asymmetry in the dominant modes of the conformational fluctuations with or without the presence of pgRNA. Furthermore, the simulations revealed that the presence of pgRNA enhances the overall mechanical stability of the virion particle. Electrostatic interactions between the disordered CTD of capsid and pgRNA were found to play a crucial role in modulating viral mechanical stability. Decreasing the electrostatic interactions by CTD phosphorylation or high salt concentration significantly reduces the mechanical stability of the HBV capsid containing pgRNA. Finally, the 2-fold symmetric sites have been proposed to be the most vulnerable to rupture during the initial stages of capsid disassembly. These findings could enhance our understanding of the physical basis of viral invasion and provide valuable insights into the development of antiviral drugs.

Large mitochondrial genomes in tenthredinid sawflies (Hymenoptera, Tenthredinidae)

We sequenced and assembled mitochondrial genomes of three tenthredinid sawflies (Euura poecilonota, E. striata, and Dolerus timidus) using Oxford Nanopore Technologies’ MinION. The Canu assembler produced circular assemblies (23,000–40,000 bp). Still, errors were found in the highly repetitive non-coding control region because of the fragmented DNA which led to no reads spanning the complete control region, preventing its reliable assembly. Based on the non-repetitive coding region’s sequencing coverage, we estimate the lengths of mitochondrial genomes of E. poecilonota, D. timidus, and E. striata to be about 30,000 bp, 31,000 bp, and 37,000 bp and control region to be 15,000 bp, 16,000 bp, and 22,000 bp respectively. All standard bilaterian mitochondrial genes are in the same order and orientation, except trnQ, which is on the minus strand in Euura and the plus strand in Dolerus. Using published tenthredinid genome data, we show that control region lengths are often underestimated.

Evolution of RNA Viruses: Reasons for the Existence of Separate Plus, Minus, and Double-Strand Replication Strategies.

Plus, minus, and double-strand RNA viruses are all found in nature. We use computational models to study the relative success of these strategies. We consider translation, replication, and virion assembly inside one cell, and transmission of virions between cells. For viruses which do not incorporate a polymerase in the capsid, transmission of only plus strands is the default strategy because virions containing minus strands are not infectious. Packaging only plus strands has a significant advantage if the number of RNA strands produced per cell is larger than the number of capsids. In this case, by not packaging minus strands, the virus produces more plus-strand virions. Therefore, plus-strand viruses are selected at low multiplicity of infection. However, at high multiplicity of infection, it is preferable to package both strands because the additional minus virions produced are helpful when there are multiple infections per cell. The fact that plus-strand viruses are widespread while viruses that package both strands are not seen in nature suggests that RNA strands are indeed produced in excess over capsids, and that the multiplicity of infection is not sufficiently high to favor the production of both kinds of virions. For double-strand viruses, we show that it is advantageous to produce only plus strands from the double strand within the cell, as is observed in real viruses. The reason for the success of minus-strand viruses is more puzzling initially. For viruses that incorporate a polymerase in the virion, minus virions are infectious. However, this is not sufficient to explain the success of minus-strand viruses, because in this case, viruses that package both strands outcompete those that package only minus or only plus. Real minus-strand viruses make use of replicable strands that are coated by a nucleoprotein, and separate translatable plus strands that are uncoated. Here we show that when there are distinct replicable and translatable strands, minus-strand viruses are selected.

Hepatitis B virus particles in serum contain minus strand DNA and degraded pregenomic RNA of variable and inverse lengths.

This study utilized digital PCR to quantify HBV RNA and HBV DNA within three regions of the HBV genome. Analysis of 75 serum samples from patients with chronic infection showed that HBV RNA levels were higher in core than in S and X regions (median 7.20 vs. 6.80 and 6.58 log copies/mL; p < .0001), whereas HBV DNA levels showed an inverse gradient (7.71 vs. 7.73 and 7.77 log copies/mL, p < .001). On average 80% of the nucleic acid was DNA by quantification in core. The core DNA/RNA ratio was associated with viral load and genotype. In individual patients, the relations between RNA levels in core, S and X were stable over time (n = 29; p = .006). The results suggest that pregenomic RNA is completely reverse transcribed to minus DNA in ≈75% of the virus particles, whereas the remaining 25% contain both RNA and DNA of lengths that reflect variable progress of the polymerase.

A selective HIV-1 RNA G-quadruplex-targeting L-aptamer-D-antisense conjugate inhibits HIV-1 minus strand transfer.

Herein, we develop an L-RNA aptamer, L-Apt.T8, for recognizing HIV-1 U3-III RNA G-quadruplex, and conjugate the L-Apt.T8 with DNA antisense (L-Apt.T8-10D) to enhance the binding affinity and selectivity. We demonstrate that L-Apt.T8-10D can inhibit HIV-1 U3-tRNA segment complementary binding and nucleocapsid interaction, and interrupt in vitro HIV-1 minus strand transfer.

The genome of a bunyavirus cannot be defined at the level of the viral particle but only at the scale of the viral population.

Bunyaviruses are enveloped negative or ambisense single-stranded RNA viruses with a genome divided into several segments. The canonical view depicts each viral particle packaging one copy of each genomic segment in one polarity named the viral strand. Several opposing observations revealed nonequal ratios of the segments, uneven number of segments per virion, and even packaging of viral complementary strands. Unfortunately, these observations result from studies often addressing other questions, on distinct viral species, and not using accurate quantitative methods. Hence, what RNA segments and strands are packaged as the genome of any bunyavirus remains largely ambiguous. We addressed this issue by first investigating the virion size distribution and RNA content in populations of the tomato spotted wilt virus (TSWV) using microscopy and tomography. These revealed heterogeneity in viral particle volume and amount of RNA content, with a surprising lack of correlation between the two. Then, the ratios of all genomic segments and strands were established using RNA sequencing and qRT-PCR. Within virions, both plus and minus strands (but no mRNA) are packaged for each of the three L, M, and S segments, in reproducible nonequimolar proportions determined by those in total cell extracts. These results show that virions differ in their genomic content but together build up a highly reproducible genetic composition of the viral population. This resembles the genome formula described for multipartite viruses, with which some species of the order Bunyavirales may share some aspects of the way of life, particularly emerging properties at a supravirion scale.

The minus strand of positive-sense RNA viruses encodes small proteins

It is widely accepted that the minus strands of positive single-strand RNA (+ssRNA) viruses function as replication templates only. Gong et al. revealed that the minus strand of two unrelated +ssRNA viruses encodes proteins. This textbook-changing discovery calls for the reconsideration of the molecular mechanisms underlying the infection cycle of +ssRNA viruses.

Integrative DNA methylome and transcriptome analysis reveals DNA adenine methylation is involved in Salmonella enterica Typhimurium response to oxidative stress.

Salmonella could survive and replicate in macrophages, where it encounters multiple stresses. Deficiency of a DNA adenine methyltransferase impairs the survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in hydrogen peroxide. To investigate whether DNA methylation is involved in the expression of oxidative stress-responsive genes, we combined RNA-seq and single-molecule real-time sequencing to integrate transcriptome and methylome analysis. Here, we show that (i) the entire amount of m6A GATC remains stable during oxidative stress; (ii) no significant association is observed between DNA methylation and transcription level in most genes; (iii) coincidence of level change between transcription and m6A GATC in the regulatory regions is identified in 49 genes under oxidative stress. Some of them are known to contribute to bacterial defenses against oxidative stress through reducing H2O2 levels, directing aberrant protein product degradation and inducing outer membrane protein expression. Specifically, the transcription level of ahpCF is negatively correlated to the m6A GATC level in its regulatory region. The transcription of smpB is elevated along with the decrease in m6A level at position 2,879,954 on the minus strand. In contrast, mRNA levels of STM14_2773 and ycfR are positively correlated with m6A GATC content in their regulatory regions. Highly stable DNA methylome and coupled change of m6A GATC with gene expression in specific positions suggest that DNA methylation homeostasis at genome-wide and plasticity in specific regions are crucial for bacterial response to oxidative stress. These findings provide new insights into the epigenetic regulatory mechanisms of gene expression in S. Typhimurium in the host microenvironment.IMPORTANCEThe intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) comes across a wide variety of stresses from entry to dissemination, such as reactive oxygen species. To adapt itself to oxidative stress, Salmonella must adopt various and complex strategies. In this study, we revealed that DNA adenine methyltransferase was essential for S. Typhimurium to survive in hydrogen peroxide. We then screened out oxidative stress-responsive genes that were potentially regulated by DNA methylation in S. Typhimurium. Our results show that the DNA methylome is highly stable throughout the genome, and the coupled change of m6A GATC with gene expression is identified in only a few positions, which suggests the complexity of the DNA methylation and gene expression regulation networks. The results may shed light on our understanding of m6A-mediated gene expression regulation in bacteria.

Trajectories of SARS-CoV-2 viral shedding among admitted patients at a tertiary-care center in California, 2020–2022

Background: SARS-CoV-2 viral load decreases over time after illness onset. However, immunocompromised patients may take longer for viral load decrease or have a more erratic viral-load trajectory. We used strand-specific assay data from admitted patients to evaluate viral-load trajectories after illness onset. Methods: We reviewed records of hospitalized patients with a positive SARS-CoV-2 PCR and tested using the strand-specific SARS-CoV-2 PCR during July 2020–April 2022. At Stanford Healthcare, we use a 2-step reverse real-time polymerase chain reaction (rRT-PCR) assay specific to the minus strand of the SARS-CoV-2 envelope gene to assess infectivity. Restricting our analysis to each patient’s first strand-specific assay, we used logistic regression models to compare patients with single versus multiple assays. Among patients with multiple tests, we compared those who had an upward trajectory in cycle threshold (Ct) values (a surrogate of decreasing viral load) versus those who did not. We analyzed presence of symptoms, immunocompromised state, immunosuppression reason, and severe COVID-19 leading to ICU care in univariate and multivariate models that further adjust for additional covariates. Significant differences were assessed using logistic regression odds ratios and an α level of 0.05. Results: In total, 848 inpatients were included. Among them, 703 were tested only once and 145 were tested 2–6 times. The longest duration of minus-strand detection was 163 days. In univariate analyses, patients with a single minus-strand assay had lower odds of being symptomatic (OR, 0.55), of being immunocompromised (OR, 0.58), and of being admitted to the ICU with severe COVID-19 (OR, 0.49). In the multivariate analysis, being admitted to the ICU with severe COVID-19 was the only significant variable associated with having &gt;1 test (OR, 2.44). Among patients who had multiple strand-specific SARS-CoV-2 assays, 119 had upward minus-strand trends of Ct values (as expected) and 26 did not. Being immunocompromised was associated with nonrising minus-strand CT values (OR, 33.3) when holding all other covariates in the model constant. Conclusions: Immunocompromised patients with COVID-19 tend to actively replicate for longer and have unexpected viral trajectories compared to immunocompetent patients. Among immunocompromised patients, suspension of transmission-based precautions may require a case-by-case evaluation.Disclosures: None

Genome-wide landscapes of genes and repeatome reveal the genomic differences between the two subspecies of peanut (Arachis hypogaea)

Distribution and structural features of genes, repeat elements and transposable elements (TEs) were studied to identify the genomic differences between the two subspecies (ssp. hypogaea and ssp. fastigiata) of peanut (Arachis hypogaea L.). A total of 67128 predicted genes, 2738666 copies of TEs and 162361 tandem repeats from the reference genome of Tifrunner were employed for this study. Of the 67128 genes, 33622 were reading on the plus strand, while 33506 were traced on the minus strand. Though B03 had the highest number of genes (4524), A08 recorded the highest density (53 genes/Mb) of genes in the genome. Telomeric regions had the highest density of genes. The average length of the genes was 3971 bp with majority of the genes (39228) containing one to five exons. The gene Arahy.11.0DU9MH had the insertion of 28 different types of TEs, and was the longest gene in the peanut genome. A total of 15731 genes were monomorphic in terms of SNPs across 179 accessions, while 7401 genes showed polymorphism at one nucleotide, indicating very low allelic variation at these genes. Remaining 66% of the genes had two or more SNPs, and therefore showed relatively high allelic variation. Among the 101 unique types of TEs, the Retro elements (869279) followed by CACTA (272596) and Mu (250248) TEs were most predominant. Telomeric regions showed less density of TEs than the regions. On an average, each gene contained 1.8 copies of TEs, and 35706 genes did not have the insertion of any TEs. A QTL-Seq approach could identify 186 SNPs and 26 gene differences between the two subspecies of A. hypogaea. Two of the 26 genes showed allelic variation in terms of SNPs and TEs.

Low infectivity among asymptomatic patients with a positive SARS-CoV-2 admission test at a tertiary-care center, 2020–2022

Background: Many hospitals have implemented admission SARS-CoV-2 testing to evaluate for the need for transmission-based precautions. However, a positive test in an asymptomatic patient may represent (1) active infection, signifying infectiousness; (2) false positivity; or (3) past infection with prolonged viral shedding. We used a strand-specific SARS-CoV-2 reverse real-time polymerase chain reaction (rRT-PCR) assay to assess infectivity among asymptomatic patients with a positive SARS-CoV-2 PCR admission test. Methods: We used a 2-step rRT-PCR specific to the minus strand of the SARS-CoV-2 envelope gene. We reviewed records of patients with a positive SARS-CoV-2 PCR who were also tested for the strand-specific SARS-CoV-2 PCR within 2 days of admission at Stanford Health Care during July 2020–April 2022. We restricted our analysis to each patient’s first test. We calculated the percentage of detectable minus strand-specific tests among asymptomatic patients over time and gathered descriptive statistics for age, sex, and immunocompromised state. Results: In total, 848 admitted patients had strand-specific SARS-CoV-2 assays performed. Of 532 patients with a strand-specific assay done within 2 days of admission, 242 (45%) were asymp tomatic. Among asymptomatic patients, the mean age was 56 years (range, 19–99), 133 (55%) were male, 50 (21%) had immunocompromising conditions, and 30 (12%) were admitted for a surgical procedure. In total, 21 (9%; range, 4%–25% per quarter) had detectable minus strand-specific assays (Fig. 1). Conclusions: Most asymptomatic patients tested for SARS-CoV-2 on admission were not infectious. Hospitals using SARS-CoV-2 PCR admission testing may need to re-evaluate the continued use of this practice.Fig. 1. Minus strand-specific SARS-CoV-2 assay percentage positivity per quarter among asymptomatic patients tested within 2 days of admission. The peak positivity in November 2021–January 2022 quarter coincided with the SARS-CoV-2 omicron variant surge in our county.Disclosure: None

A microRNA Arising from the Negative Strand of SARS-CoV-2 Genome Targets FOS to Reduce AP-1 Activity.

Virus-encoded microRNAs were first reported in the Epstein-Barr virus in 2004. Subsequently, a few hundred viral miRNAs have been identified, mainly in DNA viruses belonging to the herpesviridae family. To date, only 30 viral miRNAs encoded by RNA viruses are reported by miRBase. Since the outbreak of the SARS-CoV-2 pandemic, several studies have predicted and, in some cases, experimentally validated miRNAs originating from the positive strand of the SARS-CoV-2 genome. By integrating NGS data analysis and qRT-PCR approaches, we found that SARS-CoV-2 also encodes for a viral miRNA arising from the minus (antisense) strand of the viral genome, in the region encoding for ORF1ab, herein referred to as SARS-CoV-2-miR-AS1. Our data show that the expression of this microRNA increases in a time course analysis of SARS-CoV-2 infected cells. Furthermore, enoxacin treatment enhances the accumulation of the mature SARS-CoV-2-miR-AS1 in SARS-CoV-2 infected cells, arguing for a Dicer-dependent processing of this small RNA. In silico analysis suggests that SARS-CoV-2-miR-AS1 targets a set of genes which are translationally repressed during SARS-CoV-2 infection. We experimentally validated that SARS-CoV-2-miR-AS1 targets FOS, thus repressing the AP-1 transcription factor activity in human cells.

OBSCN restoration via OBSCN-AS1 long-noncoding RNA CRISPR-targeting suppresses metastasis in triple-negative breast cancer

Mounting evidence implicates the giant, cytoskeletal protein obscurin (720 to 870 kDa), encoded by the OBSCN gene, in the predisposition and development of breast cancer. Accordingly, prior work has shown that the sole loss of OBSCN from normal breast epithelial cells increases survival and chemoresistance, induces cytoskeletal alterations, enhances cell migration and invasion, and promotes metastasis in the presence of oncogenic KRAS. Consistent with these observations, analysis of Kaplan-Meier Plotter datasets reveals that low OBSCN levels correlate with significantly reduced overall and relapse-free survival in breast cancer patients. Despite the compelling evidence implicating OBSCN loss in breast tumorigenesis and progression, its regulation remains elusive, limiting any efforts to restore its expression, a major challenge given its molecular complexity and gigantic size (~170 kb). Herein, we show that OBSCN-Antisense RNA 1 (OBSCN-AS1), a novel nuclear long-noncoding RNA (lncRNA) gene originating from the minus strand of OBSCN, and OBSCN display positively correlated expression and are downregulated in breast cancer biopsies. OBSCN-AS1 regulates OBSCN expression through chromatin remodeling involving H3 lysine 4 trimethylation enrichment, associated with open chromatin conformation, and RNA polymerase II recruitment. CRISPR-activation of OBSCN-AS1 in triple-negative breast cancer cells effectively and specifically restores OBSCN expression and markedly suppresses cell migration, invasion, and dissemination from three-dimensional spheroids invitro and metastasis invivo. Collectively, these results reveal the previously unknown regulation of OBSCN by an antisense lncRNA and the metastasis suppressor function of the OBSCN-AS1/OBSCN gene pair, which may be used as prognostic biomarkers and/or therapeutic targets for metastatic breast cancer.

1927. Use of a SARS-CoV-2 Strand-specific Assay to evaluate for Prolonged Viral Replication among Hospitalized Patients. Tertiary Care Center, California 2020–2022

Abstract Background Determining if a patient with SARS-CoV-2 remains infectious is an infection control challenge in healthcare settings; specially, among critically ill or profoundly immunosuppressed patients. We use an assay that detects minus-strand RNA as a surrogate for actively replicating SARS-CoV-2. We report positive strand-specific assays in relationship to time since admission and describe patients with a detectable strand-specific assay &amp;gt;20 days since admission. Methods We use a 2-step rRT-PCR specific to the minus strand of the SARS-CoV-2 envelope gene. The strand-specific assay is used to evaluate for infectivity in asymptomatic patients with a positive admission screening or pre-procedural test or if ongoing replication is suspected (critical illness or profound immunosuppression). We retrieved strand-specific test results for patients hospitalized at Stanford Healthcare during August 2020–March 2022. We describe clinical characteristics for patients with a detectable minus strand-specific test &amp;gt;20 days since admission. Results A total of 774 strand-specific tests were collected from 624 hospitalized patients. A total of 523 patients had only one test (84%) and 101 (16%) had ≥2 tests. The test positivity rate varied by time since admission: 19% in tests performed 0–5 days, 28% in 6–10 days, 22% in 11–20 days, and 41% in those &amp;gt;20 days since admission. Among 35 patients tested &amp;gt;20 days since admission, 13 (37%) had ≥1 detectable minus strand-specific test. Most were male (n=8, 62%) and mean age was 59. Of 13 patients with a detectable assay, seven (54%) had prolonged viral replication with persistent symptoms and detectable minus strand assays for &amp;gt;20 days from symptom onset. Of these seven patients, four had a transplant (3 lung, 1 liver), 1 ovarian cancer, 1 CAR-T cell therapy, and 1 ESRD without immunosuppression. The remaining eight patients with a detectable assay &amp;gt;20 days since admission had illness onset while hospitalized. Conclusion Among hospitalized patients with SARS-CoV-2 infection, we found a varying positivity rate according to the timing of testing, possibly reflecting different indications for the test. The strand-specific assay may help assess for infectiousness in profoundly immunocompromised patients. Disclosures Aruna Subramanian, MD, Gilead Sciences: Grant/Research Support|Regeneron, Inc: Grant/Research Support.

1527. Use of a SARS-CoV-2 Strand-specific Assay to detect Relapse or Ongoing Infection at a Tertiary Care Center, California 2020–2022

Abstract Background Determining if a patient with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains infectious can be challenging in patients with severe disease or those profoundly immunosuppressed. We use an assay that detects minus-strand RNA as a surrogate for actively replicating SARS-CoV-2. Here, we describe a cohort of patients who had strand-assay reversion: a detectable minus strand-specific assay after having had an undetectable value which may signify relapse of infection or reinfection. Methods We used a 2-step rRT-PCR specific to the minus strand of the SARS-CoV-2 envelope gene. The strand-specific assay is used to evaluate for infectivity in asymptomatic patients with a positive screening admission or pre-procedural test or in cases when ongoing infection was suspected (critical illness or profound immunosuppression). We collected minus strand-specific assays performed at Stanford Healthcare during August 2020–April 2022. We describe basic demographics and clinical characteristics for patients who reverted from undetectable to detectable using the minus strand-specific assay. Results A total of 2,505 strand-specific tests were collected from August 2020–April 2022 from 2,064 patients. A total of 292 (14%) had two or more strand-specific tests. Of them, 19 (7%) had an undetected minus strand-specific assay followed by a subsequent detectable value. Of them, seven (37%) had a minus strand-specific CT value of &amp;lt; 33. Most were male (n=4), median age was 54 (range: 8–62). All were profoundly immunosuppressed: Four had hematologic malignancies and three were post transplantation (kidney, lung, bone marrow). Median time from onset of symptoms or first positive test to reversion was 41 days (range:27–159). Median time from undetectable to detectable minus strand specific test was 26 days (range:4–34). All cases were considered relapses or ongoing infection rather than a new infection. Conclusion Among patients with SARS-CoV-2 infection and consecutive strand-specific testing, a small proportion reverted from negative to positive. Most were profoundly immunosuppressed. The strand-specific assay can be an important tool in the evaluation of suspected cases of relapse or reinfection. Disclosures Aruna Subramanian, MD, Gilead Sciences: Grant/Research Support|Regeneron, Inc: Grant/Research Support.

Whole mitochondrial genome phylogeny of Drosophilidae

A total of 241 mitochondrial genomes were assembled and annotated from the SRA database to reconstruct a mtDNA genome phylogeny for the genus Drosophila, the family Drosophilidae, and close relatives. The resulting mtDNA genome phylogeny is largely congruent with previous higher-level analyses of Drosophila species with the exception of the relationships between the melanogaster, montium, anannassae, saltans and obscura groups. Although relationships within these species groups are congruent between nuclear and mtDNA studies, the mtDNA genome phylogeny of the groups is different when compared to earlier studies. Monophyly of known species groups within the genus Drosophila are highly supported and, as in previous work, the genera Lordiphosa, Hirtodrosophila, Zaprionus and Scaptomya are all imbedded within the genus Drosophila. Incongruence and partitioned support analyses indicate that DNA sequences are better at resolving the phylogeny than their translated protein sequences. Such analyses also indicate that genes on the minus strand of the circular molecule (Lrrna, Srrna, ND4, ND4L and ND5) provide most of the support for the overall phylogenetic hypothesis.

A Concise Review on Dysregulation of LINC00665 in Cancers

Long Intergenic Non-Protein Coding RNA 665 (LINC00665) is an RNA gene located on the minus strand of chromosome 19. This lncRNA acts as a competing endogenous RNA for miR-4458, miR-379-5p, miR-551b-5p, miR-3619-5p, miR-424-5p, miR-9-5p, miR-214-3p, miR-126-5p, miR-149-3p, miR-379-5p, miR-665, miR-34a-5p, miR-186-5p, miR-138-5p, miR-181c-5p, miR-98, miR-195-5p, miR-224-5p, miR-3619, miR-708, miR-101, miR-1224-5p, miR-34a-5p, and miR-142-5p. Via influencing expression of these miRNAs, it can enhance expression of a number of oncogenes. Moreover, LINC00665 can influence activity of Wnt/β-Catenin, TGF-β, MAPK1, NF-κB, ERK, and PI3K/AKT signaling. Function of this lncRNA has been assessed through gain-of-function tests and/or loss-of-function studies. Furthermore, diverse research groups have evaluated its expression levels in tissue samples using microarray and RT-qPCR techniques. In this manuscript, we have summarized the results of these studies and categorized them in three sections, i.e., cell line studies, animal studies, and investigations in clinical samples.

Rolling Circles as a Means of Encoding Genes in the RNA World.

The rolling circle mechanism found in viroids and some RNA viruses is a likely way that replication could have begun in the RNA World. Here, we consider simulations of populations of protocells, each containing multiple copies of rolling circle RNAs that can replicate non-enzymatically. The mechanism requires the presence of short self-cleaving ribozymes such as hammerheads, which can cleave and re-circularize RNA strands. A rolling circle must encode a hammerhead and the complement of a hammerhead, so that both plus and minus strands can cleave. Thus, the minimal functional length is twice the length of the hammerhead sequence. Selection for speed of replication will tend to reduce circles to this minimum length. However, if sequence errors occur when copying the hammerhead sequence, this prevents cleavage at one point, but still allows cleavage on the next passage around the rolling circle. Thus, there is a natural doubling mechanism that creates strands that are multiple times the length of the minimal sequence. This can provide space for the origin of new genes with beneficial functions. We show that if a beneficial gene appears in this new space, the longer sequence with the beneficial function can be selected, even though it replicates more slowly. This provides a route for the evolution of longer circles encoding multiple genes.

Novel utilization of strand‐specific reverse transcription polymerase chain reaction in perioperative clinical decision making for SARS‐CoV‐2 polymerase chain reaction positive patients

In order to prevent in‐hospital transmission and potential complications related to SARS‐CoV‐2 in the perioperative patient, most healthcare institutions require preoperative testing for SARS‐CoV‐2 prior to proceeding with elective surgery. The Centers for Disease Control and Prevention (CDC) recommends a time and symptom‐based duration of isolation for the presumed infectious period. The guidance to avoid retesting of asymptomatic patients in the 90 days following a positive reverse transcription polymerase chain reaction (RT‐PCR) test is because of the possibility of detection of non‐infectious viral shedding. When to reschedule asymptomatic patients who test RT‐PCR positive for SARS‐CoV‐2 preoperatively is of considerable debate, both from the perspective of ensuring a patient's full preoperative fitness, as well as reducing the risk of viral transmission within the hospital. We describe the novel perioperative use of a strand‐specific assay to detect minus strand ribonucleic acid (RNA) in a clinical decision‐making algorithm to determine optimal timing of elective surgery after a patient tests RT‐PCR positive for SARS‐CoV‐2. This is the first description in the literature of an attempt to further stratify patients who repeatedly test positive for SARS‐CoV‐2 into infectious versus non‐infectious for perioperative planning.

Molecular cloning of a new cry2A-type gene from Bacillus thuringiensis strain Nn10 and its expression studies

In the present study, eight indigenous Bacillus thuringiensis isolates of Western Ghats of India with more than 90% toxicity against Helicoverpa armigera were characterized for cry2A gene sub families. Seven of the eight isolates harboured cry2Aa, cry2Ab and cry2Ac genes alone and or in combination. Further, the indigenous cry2Aa gene(s) from Bacillus thuringiensis isolate Nn10 which showed 100% mortality against Helicoverpa armigera was cloned and expressed into recombinant Bt strains for management of resistance development in insects. The ORF of cry2Aa (∼1.9 kb) gene(s) from Nn10 isolate was ligated with T/A vector (pTZ57 R/T) and expressed in E. coli, DH5α. Automated sequence analysis of newly cloned recombinant cry2Aa revealed 99% homology to 916 bases in the 3′ region of minus strand and 100% homology with 720 bases in the 5’ region of holotype cry2Aa1 gene. The partial Cry2Aa amino acid sequence of Bt strain, Nn10, deduced from the nucleotide sequence generated by M13F primer showed four amino acid variation in comparison to Cry2Aa1 holotype, at 338, 345, 346 and 489th position of ORF and the sequence was submitted to the NCBI. Further the expression of ORF of cry2Aa of Nn10 into acrystalliferous Bt strain, 4Q7 using expression vector pHT3P2T under the transcriptional control of cry3Aa promoter and cry2Aa terminator. SDS PAGE analysis of recombinant protein exhibited a prominent band of about 65 kDa. Bioassay studies revealed that recombinant proteins, Cry2Aa of Nn10 was toxic to Helicoverpa armigera with LC50 value of 7.26 μg ml−1.