Full Sequence Research Articles

Allele-Specific Dual PCRs to Identify Members of the 27a Cluster of PPV.

Porcine Parvovirus (PPV) is one of the most important infectious agents causing severe reproductive failure in pigs. In the last two decades a particular, a novel genotype emerged in Europe and PPV-27a was named as the prototype of this genetic cluster. It was suggested that members of the PPV-27a cluster may adversely influence effective vaccination against PPV. For a reliable updated 27a definition, we aligned 93 databank-deposited partial or full nucleotide and protein sequences of the VP2 of different PPV isolates. We confirmed that the 27a cluster could indeed be distinguished from other members of the species, however, some divergences were identified compared to earlier defined genetic markers. Based on genetic differences, we developed a dual allele-specific polymerase chain reaction for the easy and quick discrimination of members of the 27a cluster from other PPV strains. The detection limit of dual PCR was found <1.66 × 104 copies/reaction. To sensitize and make it more user friendly, the method was further developed for qPCR application with fluorescent probes. Regarding the detection limit of the two PCRs (<1.66 × 104 copies/reaction of the dual PCR versus <2.40 × 102 copy/reaction of the dual qPCR), approximately two log improvement was achieved in the sensitivity of the method.

A new strategy: identification of specific antibodies for neutralizing epitope on SARS-CoV-2 S protein by LC-MS/MS combined with immune repertoire

Although the SARS-CoV-2 vaccine has been widely used worldwide, not all individuals can produce neutralization antibodies, so it is still urgent to find and prepare neutralization antibodies for COVID-19 prevention or treatment. In this study, we created a new strategy to effectively obtain neutralizing antibodies or complementary determining region 3 (CDR3) of neutralizing antibodies against SARS-CoV-2. We first predicted and synthesized several B cell epitopes on RBD and adjacent RBD of S protein, then the B cell epitopes were used to prepare affinity chromatography columns respectively and purify the binding IgG from serum samples of convalescent COVID-19 patients. After these IgGs were identified to have neutralizing activity, the peptide sequences of the antigen-binding regions (variable region) of neutralizing antibodies were analyzed by protein mass spectrometry. Subsequently, the B cells from the same individual were sorted and used to obtain their full BCR repertoire by 5′ RACE combined with high-throughput of PacBio sequencing method. Then, the peptide sequence of neutralizing antibody variable region by protein mass spectrometry was mapped to the full BCR repertoire and found the full variable region sequence of neutralizing antibodies. Finally, we obtained and synthesized numerous CDR3 peptides of neutralizing antibodies to confirm the neutralizing activity for SARS-CoV-2 infection. Our results indicate that the novel scheme will be suitable for rapid screening of neutralizing antibodies, including screening neutralizing antibodies against SARS-CoV-2 and other pathogenic microorganisms.

A Continuous Robot Vision Approach for Predicting Shapes and Visually Perceived Weights of Garments

We present a continuous perception approach that learns geometric and physical similarities between garments by continuously observing a garment while a robot picks it up from a table. The aim is to capture and encode geometric and physical characteristics of a garment into a manifold where a decision can be carried out, such as predicting the garment’s shape class and its visually perceived weight. Our approach features an early stop strategy, which means that a robot does not need to observe a full video sequence of a garment being picked up from a crumpled to a hanging state to make a prediction, taking 8 seconds in average to classify garment shapes. In our experiments, we find that our approach achieves prediction accuracies of 93% for shape classification and 98.5% for predicting weights and advances state-of-art approaches in similar robotic perception tasks by <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$\,22\%$</tex-math></inline-formula> for shape classification.

First Report of Fusarium oxysporum f. sp. cubense Tropical Race 4 Causing Fusarium Wilt in Cavendish Bananas in Peru.

Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), the causal agent of Fusarium wilt of banana (FWB), is currently the major threat to the banana industry worldwide (Dita et al. 2018). Restricted to South Asia for more than 20 years, Foc TR4 has spread in the last years to the Middle East, Mozambique, and Colombia (García-Bastidas et al. 2019; https://pestdisplace.org/embed/news/map/disease/11). The incursion of Foc TR4 in Colombia increased awareness and prevention efforts across Latin American and the Caribbean (LAC). However, new Foc TR4 outbreaks in LAC countries were considered a matter of time. In April 2021, banana (Musa spp., Cavendish, AAA) plants (30% of incidence) showing typical symptoms of FWB, such as leave yellowing, wilting and vascular discoloration were observed in one farm (about 1 ha) located in Querecotillo, Peru (4°43'54.84"S 80°33'45.00"W). Mycological analyses of samples (pseudostem strands) collected from 10 symptomatic plants were performed as described by Dita et al. (2010). These analyses revealed a continuous presence of fungal colonies identified as Fusarium oxysporum species complex. Molecular diagnostics targeting two different genome regions (Dita et al 2010; Li et al. 2013) identified nine of these isolates as Foc TR4. These results were further confirmed by qPCR analyses using the commercial Clear Detections TR4 kit. The genome of four single-spore isolates (PerS1, PerS2, PerS3 and PerS4) was sequenced using the Illumina platform (MiSeq Kit, 2x151 bp Paired-End). The strain PerS4 was also sequenced using Oxford Nanopore (FLOW-MIN111; R10.3 chemistry) as described by Lopez-Alvarez et al., (2020). The generated draft assembly yielded 533 contigs for a size of 47 Mbp (BioProject: PRJNA755905), which is comparable with sizes of previously reported Foc TR4 strains (Asai et al. 2019; García-Bastidas et al. 2019; Maymon et al. 2020; Warmington et al. 2019; Zheng et al. 2018). The sequence assembly showed high contiguity (94.9%) and high similarity (95.48%) with the high-quality genome sequence of the Foc TR4 isolate 'UK0001' (Warmington et al. 2019). Further analyses to identify the presence/absence of full sequences for the putative effector genes (Secreted In Xylem - SIX) and their allelic copies, also revealed that the SIX genes profile of the strains isolated from Querecotillo matched with previously reported Foc TR4 isolates (Czislowski et al. 2017). Pathogenicity tests with three isolates and water controls were performed as described by Dita et al. (2010), using five Cavendish plantlets per treatment. Four weeks after the inoculation typical external and internal symptoms of FWB were observed only in the inoculated plants. Fungal isolates recovered from inoculated plants tested positive for Foc TR4 when analyzed with PCR diagnostics as mentioned above. No fungal isolates were recovered from water-control plants which did not show any symptoms. Altogether, our results confirm the first incursion of Foc TR4 in Peru. Currently, Foc TR4 has the phytosanitary status of a present pest with restricted distribution in Peru and it is under official control of the National Plant Protection Organization - SENASA. Reinforced prevention and quarantine measures, disease monitoring and capacity building to detect, contain and manage eventual new outbreaks of Foc TR4 are strongly encouraged across LAC banana producing countries, especially for those bordering Peru with larger banana plantations, such as Ecuador and Brazil.

Tilting Modules and Exceptional Sequences for a Family of Dual Extension Algebras

We provide a classification of generalized tilting modules and full exceptional sequences for a certain family of quasi-hereditary algebras, namely dual extension algebras of path algebras of uniformly oriented linear quivers, modulo the ideal generated by paths of length two, with their opposite algebra. An important step in the classification is to prove that all indecomposable self-orthogonal modules (with respect to extensions of positive degree) admit a filtration with standard subquotients or a filtration with costandard subquotients. Furthermore, we prove that that every generalized tilting module, not equal to the characteristic tilting modules, admits either a filtration with standard subquotients or a filtration with costandard subquotients. Since the algebras studied in this article admit a simple-preserving duality, combining these two statements reduces the problem to classifying generalized tilting modules admitting a filtration with standard subquotients. To finalize the classification of generalized tilting modules we develop a combinatorial model for the poset of indecomposable self-orthogonal modules with standard filtration with respect to the relation arising from higher extensions. This model is also used for the classification of full exceptional sequences.

Accelerated cardiac T1 mapping with recurrent networks and cyclic, model-based loss.

Using the spin-lattice relaxation time (T1) as a biomarker, the myocardium can be quantitatively characterized using cardiac T1 mapping. The modified Look-Locker inversion (MOLLI) recovery sequences have become the standard clinical method for cardiac T1 mapping. However, the MOLLI sequences require an 11-heartbeat breath-hold that can be difficult for subjects, particularly during exercise or pharmacologically induced stress. Although shorter cardiac T1 mapping sequences have been proposed, these methods suffer from reduced precision. As such, there is an unmet need for accelerated cardiac T1 mapping. To accelerate cardiac T1 mapping MOLLI sequences by using neural networks to estimate T1 maps using a reduced number of T1-weighted images and their corresponding inversion times. In this retrospective study, 911 pre-contrast T1 mapping datasets from 202 subjects (128 males, 56 ± 15 years; 74 females, 54 ± 17 years) and 574 T1 mapping post-contrast datasets from 193 subjects (122 males, 57 ± 15 years; 71 females, 54 ± 17 years) were acquired using the MOLLI-5(3)3 sequence and the MOLLI-4(1)3(1)2 sequence, respectively. All acquisition protocols used similar scan parameters: , , and , gadoteridol (ProHance, Bracco Diagnostics) dose . A bidirectional multilayered long short-term memory (LSTM) network with fully connected output and cyclic model-based loss was used to estimate T1 maps from the first three T1-weighted images and their corresponding inversion times for pre- and post-contrast T1 mapping. The performance of the proposed architecture was compared to the three-parameter T1 recovery model using the same reduction of the number of T1-weighted images and inversion times. Reference T1 maps were generated from the scanner using the full MOLLI sequences and the three-parameter T1 recovery model. Correlation and Bland-Altman plots were used to evaluate network performance in which each point represents averaged regions of interest in the myocardium corresponding to the standard American Heart Association 16-segment model. The precision of the network was examined using consecutively repeated scans. Stress and rest pre-contrast MOLLI studies as well as various disease test cases, including amyloidosis, hypertrophic cardiomyopathy, and sarcoidosis were also examined. Paired t-tests were used to determine statistical significance with . Our proposed network demonstrated similar T1 estimations to the standard MOLLI sequences (pre-contrast: vs. with ; post-contrast: vs. with ). The precision of standard MOLLI sequences was well preserved with the proposed network architecture ( vs. ). Network-generated T1 reactivities are similar to stress and rest pre-contrast MOLLI studies ( vs. with ). Amyloidosis T1 maps generated using the proposed network are also similar to the reference T1 maps (pre-contrast: vs. with ; post-contrast: vs. with ). A bidirectional multilayered LSTM network with fully connected output and cyclic model-based loss was used to generate high-quality pre- and post-contrast T1 maps using the first three T1-weighted images and their corresponding inversion times. This work demonstrates that combining deep learning with cardiac T1 mapping can potentially accelerate standard MOLLI sequences from 11 to 3 heartbeats.

A new perspective on the evolution and diversity of the genus Amdoparvovirus (family Parvoviridae) through genetic characterization, structural homology modeling, and phylogenetics.

Amdoparvoviruses (genus Amdoparvovirus, family Parvoviridae) are primarily viruses of carnivorans, but recent studies have indicated that their host range might also extend to rodents and chiropterans. While their classification is based on the full sequence of the major nonstructural protein (NS1), several studies investigating amdoparvoviral diversity have been focused on partial sequences, leading to difficulties in accurately determining species demarcations and leaving several viruses unclassified. In this study, while reporting the complete genomic sequence of a novel amdoparvovirus identified in an American mink (British Columbia amdoparvovirus, BCAV), we studied the phylogenetic relationships of all amdoparvovirus-related sequences and provide a comprehensive reevaluation of their diversity and evolution. After excluding recombinant sequences, phylogenetic and pairwise sequence identity analyses allowed us to define fourteen different viruses, including the five currently classified species, BCAV, and four additional viruses that fulfill the International Committee on Taxonomy of Viruses criteria to be classified as species. We show that the group of viruses historically known as Aleutian mink disease virus (species Carnivore amdoparvovirus 1) should be considered as a cluster of at least four separate viral species that have been co-circulating in mink farms, facilitating the occurrence of inter-species recombination. Genome organization, splicing donor and acceptor sites, and protein sequence motifs were surprisingly conserved within the genus. The sequence of the major capsid protein virus protein 2 (VP2) was significantly more conserved between and within species compared to NS1, a phenomenon possibly linked to antibody-dependent enhancement (ADE). Homology models suggest a remarkably high degree of conservation of the spikes located near the icosahedral threefold axis of the capsid, comprising the surface region associated with ADE. A surprisingly high number of divergent amino acid positions were found in the luminal threefold and twofold axes of the capsid, regions of hitherto unknown function. We emphasize the importance of complete genome analyses and, given the marked phylogenetic inconsistencies across the genome, advise to obtain the complete coding sequences of divergent strains. Further studies on amdoparvovirus biology and structure as well as epidemiological and virus discovery investigations are required to better characterize the ecology and evolution of this important group of viruses.

Comprehensive Analysis of Congenital Adrenal Hyperplasia Using Long-Read Sequencing.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that has been included in newborn screening programs. Current approaches to gene testing for CAH are facing challenges because of the complexity of the CYP21A2 locus and genetic heterogeneity of the disease. A comprehensive analysis of CAH (CACAH) combining long-range locus-specific PCR and long-read sequencing (LRS) was developed to perform full sequence analysis of 5 common CAH candidate genes, including CYP21A2, CYP11B1, CYP17A1, HSD3B2, and StAR. In a blind retrospective study, the clinical utility of CACAH was evaluated in 37 samples by comparing to standard CAH testing using multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing. Of the 37 clinical samples, a total of 69 pathogenic variants were identified, comprising 65 CYP21A2 variants, 2 HSD3B2 variants, and 2 CYP17A1 variants. For CYP21A2, the most frequent variant was c.518T > A (29.2%), followed by c.293-13C/A > G (21.5%). Compared with the current CAH testing using MLPA plus Sanger sequencing, the CACAH assay showed 100% specificity and 100% sensitivity, and precisely determined the junction sites of deletions/insertions and cis-trans configuration of multiple variants without analyzing family samples. Moreover, CACAH identified a case carrying 2 copies of CYP21A1 with the c.1451_1452delinsC variant on the same chromosome, which was not confirmed by MLPA plus Sanger sequencing. LRS-based CACAH can determine all genotypes of CAH accurately and reliably in one assay, presenting a comprehensive approach for CAH genetic diagnosis and carrier screening.

Abstract 3: Developing a translational tool for GLP-1-based therapeutic agonists in humanized GLP1R mice

Abstract Glucagon-like peptide-1 (GLP1) is a pleiotropic hormone released from gut enteroendocrine cells following nutrient ingestion. In addition to promoting insulin secretion and inhibiting glucagon secretion, GLP1 acts via the glucagon-like peptide-1 receptor (GLP1R) to reduce gastric emptying and food intake. Therefore, development of GLP1R agonists appears to be a promising therapeutic option for type 2 diabetes. To evaluate the efficacy of GLP1 analogs in preclinical studies, Biocytogen has successfully generated a novel humanized GLP1R (B-hGLP1R) knock-in mouse line to support related drug development studies. In this mouse model, the full coding sequence of the human GLP1R gene was inserted into the mouse Glp1r gene locus. Human GLP1R mRNA expression was detected by RT-PCR in B-hGLP1R mice but not in wild-type mice. Protein expression of human GLP1R in B-hGLP1R mice was confirmed by western blot analysis and immunohistochemistry using pancreatic tissues. Furthermore, flow cytometry analysis of leukocyte subpopulations showed that B-hGLP1R mice do not exhibit a change in the overall development, differentiation, or distribution of immune cell types in the spleen, blood, and lymph nodes. Additionally, our in vivo efficacy study confirmed Dulaglutide, a GLP1 receptor agonist, reduced the non-fasting blood glucose, fasting blood glucose, glucagon, and food intake levels in B-hGLP1R obese mice and increased plasma insulin and GLP1 secretion. In B-hGLP1R mice, glucose tolerance (GTT) was also improved by Dulaglutide treatment, similar GTT results were observed after treatment with PF-06882961, a phase II agent specifically recognizing human GLP1R. Altogether, B-hGLP1R mice provide an efficacious preclinical animal model to evaluate novel GLP1 based therapeutic drugs. Keywords: Humanized mice, GLP1R, agonist Citation Format: Yuelei Shen, Jiawei Yao, Mostafa Kokabee, Yanan Guo, Yang BaI, Chengzhang Shang. Developing a translational tool for GLP-1-based therapeutic agonists in humanized GLP1R mice [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 3.

Abstract 1643: B-hIL12A/hIL12B/hIL12RB1/hIL12RB2: A novel animal model for generation of IL12 therapies

Abstract The pro-inflammatory cytokine, interleukin 12 (IL12), is primarily responsible for cell-mediated immunity and is chiefly produced by antigen presenting cells. Prior preclinical studies elucidated a promising role for IL12 mediating anti-tumor effects, however, clinical studies using cancer patients showed disappointing efficacy results, as well as severe adverse effects, with administration of systemic IL12 therapies. Intriguingly, progress in targeted delivery to the tumor microenvironment renewed high expectations on IL12 as a promising anti-tumor drug either alone or in combination due to its ability to inhibit and/or reprogram immunosuppressive cell subsets. Therefore, to evaluate the efficacy of IL12 and IL12 analogs in preclinical studies, Biocytogen has successfully generated a novel hIL12A/hIL12B/hIL12RB1/hIL12RB2 knock-in mouse strain. In this mouse model, the full coding sequences (CDS) of the mouse Il12a or Il12b gene were replaced by their human counterpart IL12A or IL12B gene sequences respectively. In addition, chimeric CDS, composed of human IL12RB1 or IL12RB2 extracellular region plus mouse Il12rb1 or Il12rb2 cytoplasmic region, was inserted into the mouse endogenous gene locus in in these animals, to express an IL12 receptor (IL12R) capable of recognizing human IL12 signaling. Expression analysis by RT-PCR detected human IL12A/IL12B/IL12RB1/IL12RB2 mRNA in humanized mice but not in wild-type mice. Additionally, human IL12 p70 was successfully detected in the serum of humanized mice by ELISA. The immune cell subpopulations in spleen, blood, and lymph node did not differ between humanized mice and wild-type mice suggesting no impact on cell differentiation and maturation. Anti-tumor activity of a modified human IL12 was assessed in humanized mice bearing colon cancer MC38 cells and showed tumor inhibitory effects. Moreover, the tumor growth recovered when the administration of the drug was stopped. Altogether, B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 humanized mice provide a promising preclinical efficacy model for human IL12 therapy alone or in combination. Keywords: IL12, humanized mice, antibody efficacy Citation Format: Linlin Wang, Chang Liu, Veronika Chromikova, Ruili Lyu, Tianquan Jin, Mari Kuraguchi. B-hIL12A/hIL12B/hIL12RB1/hIL12RB2: A novel animal model for generation of IL12 therapies [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 1643.

Vectorial folding of telomere overhang promotes higher accessibility.

Human telomere overhang composed of tandem repeats of TTAGGG folds into G-quadruplex (G4). Unlike in an experimental setting in the test tube in which the entire length is allowed to fold at once, inside the cell, the overhang is expected to fold as it is synthesized directionally (5′ to 3′) and released segmentally by a specialized enzyme, the telomerase. To mimic such vectorial G4 folding process, we employed a superhelicase, Rep-X which can unwind DNA to release the TTAGGG repeats in 5′ to 3′ direction. We demonstrate that the folded conformation achieved by the refolding of full sequence is significantly different from that of the vectorial folding for two to eight TTAGGG repeats. Strikingly, the vectorially folded state leads to a remarkably higher accessibility to complementary C-rich strand and the telomere binding protein POT1, reflecting a less stably folded state resulting from the vectorial folding. Importantly, our study points to an inherent difference between the co-polymerizing and post-polymerized folding of telomere overhang that can impact telomere architecture and downstream processes.

Influence of welding sequences on induced residual stress and distortion in pipes

A three-dimensional (3D) finite element (FE) model based on thermal-elastic-plastic approach in SYSWELD® was employed to investigate the effects of welding sequences on induced residual stresses and radial distortion in AISI 316L stainless steel welded pipes. To mesh the model, the HYPERMESH® processor was utilized and the model was then exported to SYSWELD® for further thermo-mechanical analysis. The Goldak's double ellipsoidal moving heat source model was employed in SYSWELD® to obtain the volumetric heat flux density in the Gas Metal Arc Welding (GMAW) simulation process. A neural-network program was suitably trained and used to predict the parameters of Goldak's model. The obtained numerical models using predicted Goldak’s parameters were then successfully validated by experimental results in terms of size of welding pool and the temperature distribution. Four different welding sequences (concepts) were investigated and compared: three quarters and one full circumferential welding. These welding sequences were anticipated to create the least residual stress and distortion in pipe welding. The results demonstrate that the welding sequences significantly influence induced residual stresses and distortions in magnitude and in distribution. For the full circumferential welding concept, which was proposed in this study, the residual stresses and distortion present more uniform distribution along the circumferential direction than those in quarter welding sequences. The magnitude of welding distortion was also significantly lower in the full circumferential concept when compared to the quarter welding concepts. The FE results of residual stresses in the full circumferential welding sequences were accurately validated by experimental tests.

Resectable pancreatic adenocarcinoma neo-adjuvant FOLF(IRIN)OX-based chemotherapy: A multicenter, non-comparative, randomized, phase II trial (PANACHE01-PRODIGE48 study).

4134 Background: Despite the limited number of published RCTs, patients with resectable pancreatic adenocarcinoma (rPAC; NCCN criteria) seem to benefit from neoadjuvant (NAT) with regards to R0 resection rate, downstaging and survivals (PREOPANC-1; Preop-02/JSAP-05; NEONAX, SWOG05-15). Few prospective results are currently available concerning the completion of the full therapeutic sequence and oncological results. Methods: In the PANACHE01-PRODIGE48 prospective multicenter controlled non-comparative Phase II trial, 153 patients with rPDAC were randomized 2:2:1 to 4 cycles of NAT chemotherapy (mFOLFIRINOX; Arm1 or FOLFOX; Arm2) or upfront surgery (Ctrl_Arm) in 28 French centers from 02/2017 to 07/2020. Primary objective was to evaluate the safety and efficacy of two regimens of NAT chemotherapy. The main co-primary endpoints were 1y-OS rate after randomization and the rate of patients undergoing the full therapeutic sequence. Event-free survival (EFS) was used to evaluate the time to recurrence between groups. An event being defined as progression before surgery, unresectable or metastatic disease at surgical exploration, recurrence after surgery or death. Results: Of the 153 randomized patients, 146 were available for analysis (Arm1,n=70; Arm2,n=50; Ctrl_arm,n=26). In the Arm1 and 2, completion of the 4 planed cycles was 88.6 and 84%, respectively, with a dose reduction in 52% and 24%. The cumulative rates of grade 3/4/5 toxicity were 56.7 and 57.4%, respectively. The resection rates (RR) were 74, 68 and 81% respectively in Arm1, Arm2 and Ctrl_Arm. Respectively, 88.4, 91 and 85.7% of patients started adjuvant chemo. 1y-OS rates were respectively of 84.1, 71.8 and 80.8% in Arm1, Arm2 and Ctrl_Arm with a median follow-up in each group of 25.6, 33, 30.9 months. Following the intermediate analysis, the Arm2 was stopped for lack of efficacy (rejection of the H0 hypothesis for 1yOS). 1y-Event free survival (EFS) rates were 51.4% in Arm1, 43.1% in Arm2 and 41.7% in Ctrl_Arm, with corresponding median EFS of 12.4, 11 and 9.2 months. Conclusions: The feasibility and efficacy of the mFOLFIRINOX neoadjuvant chemotherapy is confirmed regarding completion of therapeutic sequence and oncological outcomes. These results confirm the rationale for ongoing clinical trials, PREOPANC3 and Alliance AO21806. Clinical trial information: NCT02959879.

Molecular Characterization of Recently Classified Newcastle Disease Virus Genotype VII.1.1 Isolated from Egypt

Newcastle disease virus (NDV) is classified as Avian OrthoAvulavirus-1 (AOAV-1) and is still one of the most important poultry diseases in the world. Collected tissue specimens (trachea, cecal tonsils, and spleen) from 85 chicken flocks (broilers, layers, and native breeds) raised in 5 Egyptian governorates (El-Qalubia, El-Behaira, El-Menofia, El-Gharbia, and Alexandria) in the period between March 2019 to February 2020. The flocks had a history of vaccination against ND and showed clinical signs and lesions of infection. We characterized the isolates molecularly using conventional RT-PCR via amplifying the NDV full Fusion protein gene. The analysis of the full F-gene sequence revealed that our isolates were classified as genotype VII with the characteristic amino acids motif in the fusion protein cleavage site for the velogenic NDV (vNDV) strains. Among the molecularly identified NDV Genotype VII isolates, 5 representative isolates in different 5 Egyptian Governorates were sequenced for full fusion protein gene (sequence). The obtained sequences were submitted on GenBank: MW580389, MW580388, MW580387, MW566177, and MW590306. The five isolates' full-length F protein gene amino acid sequences revealed 99.5% identity with the Reference GenBank NDV/Egypt/ch/MN51/2019 strain isolate. The deduced amino acids sequences' comparative alignment of the five Avian OrthoAvulavirus-1 isolates confirmed the presence of motif of virulent strains (RRQKRF) between 112 and 117 residues of fusion protein cleavage site. All five AOAV-1 isolates from 2019 and 2020 belonged to the NDV genotype classified as genotype VII.1.1 depending on full Fusion gene sequences; those five isolates were grouped with subgenotype VIId. Through significant genetic changes in vNDV observed in our study, continuous monitoring of the multiple vNDV outbreaks in vaccinated chicken and targeted evaluation of the used vaccines.

Other-Initiated Self-Repairs in Student-Student Interaction: The Frequency of Occurrence and Mechanism

The current study is intended to explore the typology of other-initiated self-repair in term of its frequency of occurrence and to investigate the mechanism of self-repair in an academic setting. To obtain the required data, a classroom conversational analysis was conducted in which the researchers observed a group of graduate students (19 active EFL speakers) in a state university in East Java, Indonesia. Based on the qualitative data analysis, the results of the study revealed that all typologies of other-initiated selfrepair were used depending on the aims the interlocutors try to gain since all typologies have diverse aims and ways. Of all typologies, confirming checks were dominantly used by the participants. Regarding the mechanisms, three types of mechanisms were found; full two-cycle repair, background check, and short-cut repair sequence.

Other-Initiated Self-Repairs in Student-Student Interaction: The Frequency of Occurrence and Mechanism

The current study is intended to explore the typology of other-initiated self-repair in term of its frequency of occurrence and to investigate the mechanism of self-repair in an academic setting. To obtain the required data, a classroom conversational analysis was conducted in which the researchers observed a group of graduate students (19 active EFL speakers) in a state university in East Java, Indonesia. Based on the qualitative data analysis, the results of the study revealed that all typologies of other-initiated selfrepair were used depending on the aims the interlocutors try to gain since all typologies have diverse aims and ways. Of all typologies, confirming checks were dominantly used by the participants. Regarding the mechanisms, three types of mechanisms were found; full two-cycle repair, background check, and short-cut repair sequence.

Reconstruction of full-length LINE-1 progenitors from ancestral genomes.

Sequences derived from the Long INterspersed Element-1 (L1) family of retrotransposons occupy at least 17% of the human genome, with 67 distinct subfamilies representing successive waves of expansion and extinction in mammalian lineages. L1s contribute extensively to gene regulation, but their molecular history is difficult to trace, because most are present only as truncated and highly mutated fossils. Consequently, L1 entries in current databases of repeat sequences are composed mainly of short diagnostic subsequences, rather than full functional progenitor sequences for each subfamily. Here, we have coupled 2 levels of sequence reconstruction (at the level of whole genomes and L1 subfamilies) to reconstruct progenitor sequences for all human L1 subfamilies that are more functionally and phylogenetically plausible than existing models. Most of the reconstructed sequences are at or near the canonical length of L1s and encode uninterrupted ORFs with expected protein domains. We also show that the presence or absence of binding sites for KRAB-C2H2 Zinc Finger Proteins, even in ancient-reconstructed progenitor L1s, mirrors binding observed in human ChIP-exo experiments, thus extending the arms race and domestication model. RepeatMasker searches of the modern human genome suggest that the new models may be able to assign subfamily resolution identities to previously ambiguous L1 instances. The reconstructed L1 sequences will be useful for genome annotation and functional study of both L1 evolution and L1 contributions to host regulatory networks.

Genome and Phylogenetic Analysis of Cucumber Green Mottle Mosaic Virus Global Isolates and Validation of a Highly Sensitive RT-qPCR Assay.

The last two decades have seen exponential growth in the international movement of seeds for annual food crops, from a gross U.S. import value of $349 million in 1999 to $1.05 billion in 2019. This has led to the proportionate growth of seedborne pathogens dispersed with seed stocks. One such viral pathogen is cucumber green mottle mosaic virus (CGMMV), a tobamovirus that infects cucurbit crops such as melon, watermelon, cucumber, pumpkin, and squash. The first CGMMV introduction to California occurred in 2013, with subsequent annual outbreaks or detections since then. Here, we describe the use of next-generation sequencing to characterize the full genomes of 25 CGMMV isolates collected between 2013 and 2020 in California, either from CGMMV field detections or seed lots identified as CGMMV positive. We sequenced an additional 31 CGMMV isolates collected in Europe, Israel, and southeast Asia that were provided by industry collaborators. We also performed an in silico nucleotide database search in GenBank for full genome CGMMV sequences to include in all in silico analyses. Based on conserved regions within the coat protein gene, we then developed a quantitative reverse-transcription PCR assay for the sensitive and specific detection of CGMMV in seed and plant samples. Finally, based on our sequence and phylogenetic analysis, our data support that CGMMV has been introduced multiple times into California.

A programmable DNA roadblock system using dCas9 and multivalent target sites.

A protein roadblock forms when a protein binds DNA and hinders translocation of other DNA binding proteins. These roadblocks can have significant effects on gene expression and regulation as well as DNA binding. Experimental methods for studying the effects of such roadblocks often target endogenous sites or introduce non-variable specific sites into DNAs to create binding sites for artificially introduced protein roadblocks. In this work, we describe a method to create programmable roadblocks using dCas9, a cleavage deficient mutant of the CRISPR effector nuclease Cas9. The programmability allows us to custom design target sites in a synthetic gene intended for in vitro studies. These target sites can be coded with multivalency-in our case, internal restriction sites which can be used in validation studies to verify complete binding of the roadblock. We provide full protocols and sequences and demonstrate how to use the internal restriction sites to verify complete binding of the roadblock. We also provide example results of the effect of DNA roadblocks on the translocation of the restriction endonuclease NdeI, which searches for its cognate site using one dimensional diffusion along DNA.

Molecular-genetic structure of &lt;i&gt;Pseudomonas phage&lt;/i&gt; BIM BV-45 D

Full nucleotide sequence of bacteriophage Pseudomonas phage BIM BV-45 D – active component of biopesticide Multiphage intended for control of crop bacterial diseases caused by Pseudomonas syringae was analyzed. It was found that phage genome is represented by linear double-stranded DNA sized 40383 b. p. (average GC contents equals 58 %), comprising 46 open reading frames, including 13 described in genomes of closely related phages. 4 control sequences typical for bacterial genes recognized by sigma factor (σ70) of RNA polymerase were detected, specific phage promoters were not localized. The identity was established of the majority of protein amino acid sequences of the Pseudomonas phage BIM BV-45 D with the proteins of the known Pseudomonas phage Andromeda (95‒100 %), at the same time, the sequences of the DNA endonuclease protein (gene 22) are similar (63 %) to the corresponding protein of Pseudomonas phage PollyC. The obtained data suggest that the mosaic structure of Pseudomonas phage BIM BV-45 D genome is due to recombinant rearrangements between the afore-mentioned phages.