One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse genetics system.

Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.

High-Complexity One-Pot Golden Gate Assembly.

Golden Gate Assembly is a flexible method of DNA assembly and cloning that permits the joining of multiple fragments in a single reaction through predefined connections. The method depends on cutting DNA using a Type IIS restriction enzyme, which cuts outside its recognition site and therefore can generate overhangs of any sequence while separating the recognition site from the generated fragment. By choosing compatible fusion sites, Golden Gate permits the joining of multiple DNA fragments in a defined order in a single reaction. Conventionally, this method has been used to join five to eight fragments in a single assembly round, with yield and accuracy dropping off rapidly for more complex assemblies. Recently, we demonstrated the application of comprehensive measurements of ligation fidelity and bias data using data-optimized assembly design (DAD) to enable a high degree of assembly accuracy for very complex assemblies with the simultaneous joining of as many as 52 fragments in one reaction. Here, we describe methods for applying DAD principles and online tools to evaluate the fidelity of existing fusion site sets and assembly standards, selecting new optimal sets, and adding fusion sites to existing assemblies. We further describe the application of DAD to divide known sequences at optimal points, including designing one-pot assemblies of small genomes. Using the T7 bacteriophage genome as an example, we present a protocol that includes removal of native Type IIS sites (domestication) simultaneously with parts generation by PCR. Finally, we present recommended cycling protocols for assemblies of medium to high complexity (12-36 fragments), methods for producing high-quality parts, examples highlighting the importance of DNA purity and fragment stoichiometric balance for optimal assembly outcomes, and methods for assessing assembly success. © 2023 New England Biolabs, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Assessing the fidelity of an overhang set using the NEBridge Ligase Fidelity Viewer Basic Protocol 2: Generating a high-fidelity overhang set using the NEBridge GetSet Tool Alternate Protocol 1: Expanding an existing overhang set using the NEBridge GetSet Tool Basic Protocol 3: Dividing a genomic sequence with optimal fusion sites using the NEBridge SplitSet Tool Basic Protocol 4: One-pot Golden Gate Assembly of 12 fragments into a destination plasmid Alternate Protocol 2: One-pot Golden Gate Assembly of 24+ fragments into a destination plasmid Basic Protocol 5: One-pot Golden Gate Assembly of the T7 bacteriophage genome from 12+ parts Support Protocol 1: Generation of high-purity amplicons for assembly Support Protocol 2: Cloning assembly parts into a holding vector Support Protocol 3: Quantifying DNA concentration using a Qubit 4 fluorometer Support Protocol 4: Visualizing large assemblies via TapeStation Support Protocol 5: Validating phage genome assemblies via ONT long-read sequencing.

Chiral Ising Gross-Neveu Criticality of a Single Dirac Cone: A Quantum Monte Carlo Study.

We perform large-scale quantum Monte Carlo simulations of SLAC fermions on a two-dimensional square lattice at half filling with a single Dirac cone with N=2 spinor components and repulsive on-site interactions. Despite the presence of a sign problem, we accurately identify the critical interaction strength U_{c}=7.28±0.02 in units of the hopping amplitude, for a continuous quantum phase transition between a paramagnetic Dirac semimetal and a ferromagnetic insulator. Using finite-size scaling, we extract the critical exponents for the corresponding N=2 chiral Ising Gross-Neveu universality class: the inverse correlation length exponent ν^{-1}=1.19±0.03, the order parameter anomalous dimension η_{ϕ}=0.31±0.01, and the fermion anomalous dimension η_{ψ}=0.136±0.005.

Rewritable two-dimensional DNA-based data storage with machine learning reconstruction.

DNA-based data storage platforms traditionally encode information only in the nucleotide sequence of the molecule. Here we report on a two-dimensional molecular data storage system that records information in both the sequence and the backbone structure of DNA and performs nontrivial joint data encoding, decoding and processing. Our 2DDNA method efficiently stores images in synthetic DNA and embeds pertinent metadata as nicks in the DNA backbone. To avoid costly worst-case redundancy for correcting sequencing/rewriting errors and to mitigate issues associated with mismatched decoding parameters, we develop machine learning techniques for automatic discoloration detection and image inpainting. The 2DDNA platform is experimentally tested by reconstructing a library of images with undetectable or small visual degradation after readout processing, and by erasing and rewriting copyright metadata encoded in nicks. Our results demonstrate that DNA can serve both as a write-once and rewritable memory for heterogenous data and that data can be erased in a permanent, privacy-preserving manner. Moreover, the storage system can be made robust to degrading channel qualities while avoiding global error-correction redundancy.

Expanding the Molecular Alphabet of DNA-Based Data Storage Systems with Neural Network Nanopore Readout Processing.

DNA is a promising next-generation data storage medium, but challenges remain with synthesis costs and recording latency. Here, we describe a prototype of a DNA data storage system that uses an extended molecular alphabet combining natural and chemically modified nucleotides. Our results show that MspA nanopores can discriminate different combinations and ordered sequences of natural and chemically modified nucleotides in custom-designed oligomers. We further demonstrate single-molecule sequencing of the extended alphabet using a neural network architecture that classifies raw current signals generated by Oxford Nanopore sequencers with an average accuracy exceeding 60% (39× larger than random guessing). Molecular dynamics simulations show that the majority of modified nucleotides lead to only minor perturbations of the DNA double helix. Overall, the extended molecular alphabet may potentially offer a nearly 2-fold increase in storage density and potentially the same order of reduction in the recording latency, thereby enabling new implementations of molecular recorders.

A finite-time adaptive order estimation approach for non-integer order nonlinear systems.

This paper proposes some methods for estimating the order of nonlinear systems having non-integer order. First, the stability of time-varying order systems is studied. Afterward, the multivariable systems with time-varying incommensurate order are studied and an estimation scheme is proposed to approximate the order. In the next step, considering the pseudo-states of a system to be unknown, an order/pseudo-state estimator is designed for a category of nonlinear systems. It is shown that the method is extendible to form order/pseudo-state estimators for other classes of nonlinear systems using other traditional nonlinear observers. One of the advantages of the proposed methods is that a compact time interval is enough to guarantee bounded estimation error.

Detection and distribution of zoonotic pathogens in wild Norway rats (Rattus norvegicus) from Tehran, Iran.

This is the first study on the prevalence of vector-borne zoonotic pathogens found in Rattus norvegicus (R. norvegicus) in urban areas of Tehran, Iran. Serological tests were used to detect IgG antibodies against Coxiella burnetii (C. burnetii) and Rickettsia spp. using a commercial qualitative rat ELISA kit. The frequency of Streptobacillus moniliformis (S. moniliformis) and Bartonella spp. was determined using a conventional PCR method. Molecular detection and characterization of Leptospira spp. were conducted using TaqMan real-time PCR based on lipL32 gene and SecY typing methods. A total of 100 R. norvegicus rats were collected from five regions in Tehran, Iran, and investigated to determine their zoonotic pathogens. S. moniliformis and Bartonella spp. were detected in 23 of 100 (23%) and 17 of 100 (17%) R. norvegicus populations, respectively. The highest prevalence of S. moniliformis and Bartonella spp. with similar frequency rates (n = 6/20; 30%) was seen among the R. norvegicus rats captured from the northern and southern parts of Tehran, respectively. Seroreactivity against C. burnetii and Rickettsia spp. was detected in 4% and 1% of R. norvegicus, respectively. C. burnetii. was identified only in one rat captured from the eastern part of Tehran. Results showed that Leptospira spp. was detected only in two rats, collected from the southern part (n = 2/20; 10%) of Tehran. The secY typing method identified two different Leptospira species including L. interrogans and L. kirschneri. The results showed that urban rats might play an important role in transmission of zoonotic pathogens to humans.

Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination.

Direct cloning represents the most efficient strategy to access the vast number of uncharacterized natural product biosynthetic gene clusters (BGCs) for the discovery of novel bioactive compounds. However, due to their large size, repetitive nature, or high GC-content, large-scale cloning of these BGCs remains an overwhelming challenge. Here, we report a scalable direct cloning method named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) which consists of Cas12a digestion, a DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization. We apply this method to clone 47 BGCs ranging from 10 to 113 kb from both Actinomycetes and Bacilli with ~100% efficiency. Heterologous expression of cloned BGCs leads to the discovery of 15 previously uncharacterized natural products including six cyclic head-to-tail heterodimers with a unique 5/6/6/6/5 pentacyclic carbon skeleton, designated as bipentaromycins A-F. Four of the bipentaromycins show strong antimicrobial activity to both Gram-positive and Gram-negative bacteria such as methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus faecium, and bioweapon Bacillus anthracis. Due to its robustness and efficiency, our direct cloning method coupled with heterologous expression provides an effective strategy for large-scale discovery of novel natural products.

DNA punch cards for storing data on native DNA sequences via enzymatic nicking.

Synthetic DNA-based data storage systems have received significant attention due to the promise of ultrahigh storage density and long-term stability. However, all known platforms suffer from high cost, read-write latency and error-rates that render them noncompetitive with modern storage devices. One means to avoid the above problems is using readily available native DNA. As the sequence content of native DNA is fixed, one can modify the topology instead to encode information. Here, we introduce DNA punch cards, a macromolecular storage mechanism in which data is written in the form of nicks at predetermined positions on the backbone of native double-stranded DNA. The platform accommodates parallel nicking on orthogonal DNA fragments and enzymatic toehold creation that enables single-bit random-access and in-memory computations. We use Pyrococcus furiosus Argonaute to punch files into the PCR products of Escherichia coli genomic DNA and accurately reconstruct the encoded data through high-throughput sequencing and read alignment.

Attosecond pulse generation from $H_2

The behavior of the high-order harmonics and output attosecond pulses from hydrogen molecule ions with various internuclear distances that are exposed to high intensity incoming pulses are investigated. The incoming pulses that are spectrally wide yield from a superposition of monochromatic beams with a constant frequency distance. Our simulations show that the most intense and shortest attosecond pulses can result from hydrogen molecular ions with large internuclear distances which are exposed to irradiation of intense pulses with a frequency width greater than 0.03 a.u.

Andreev-Coulomb Drag in Coupled Quantum Dots.

The Coulomb drag effect has been observed as a tiny current induced by both electron-hole asymmetry and interactions in normal coupled quantum dot devices. In the present work we show that the effect can be boosted by replacing one of the normal electrodes by a superconducting one. Moreover, we show that at low temperatures and for sufficiently strong coupling to the superconducting lead, the Coulomb drag is dominated by Andreev processes, is robust against details of the system parameters, and can be controlled with a single gate voltage. This mechanism can be distinguished from single-particle contributions by a sign inversion of the drag current.

Severe maternal morbidity among women with a history of cesarean section at a tertiary referral teaching hospital in the southeast of Iran.

OBJECTIVES: Although the cesarean section (C-section) is known as a lifesaving procedure, it can also increase the risk of maternal mortality and morbidity. This study was conducted to achieve two objectives: (1) determination of maternal near miss (MNM) indicators and pregnancy outcomes among women with a history of C-section and (2) investigation of the women's experience of near miss events during childbirth to provide a deeper understanding of the weaknesses of care delivery process. STUDY DESIGN: The present study was conducted using a prospective cross-sectional study using the World Health Organization (organ system dysfunction-based) criteria. METHODS: All subjects were women at more than 22 weeks of gestation who gave birth in Ali ibn Abi Talib Hospital, Zahedan, Iran, and were referred to this hospital for childbirth or those within 42 days of pregnancy termination. Logistic regression was used to predict the risk factors for severe maternal outcomes (SMOs). RESULTS: Nearly 58% of all MNM cases were related to women with a history of C-section. The MNM ratio was estimated at 8.04 per 1000 live births. Moreover, the frequent causes of SMOs were reported as severe hemorrhage (58.3%), severe pre-eclampsia/eclampsia (27.8%), medical condition (8.3%), and sepsis or severe systemic infection (5.6%). Abnormally invasive placenta (61%) was the most common cause of severe hemorrhage. After adjustment for potential confounder variables, the hemoglobin level of <11 g/dl (odds ratio [OR] = 0.18, 95% confidence interval [CI] = 0.09-0.36) and place of residence (OR = 0.41, 95% CI = 0.19-0.90) remained as independent risk factors for SMOs in the final model of multivariate analysis. CONCLUSION: A risk assessment system is needed to diagnose and manage the risk factors for SMOs during prenatal care. In addition, there should be a continuous audit of the indication and number of C-sections performed across health facilities.

The clinical evaluation of platelet-rich plasma on free gingival graft's donor site wound healing.

OBJECTIVE: It has been proved that platelet-rich plasma (PRP) can promote wound healing. In this way, PRP can be advantageous in periodontal plastic surgeries, free gingival graft (FGG) being one such surgery. MATERIALS AND METHODS: In this randomized split-mouth controlled trial, 10 patients who needed bilateral FGG were selected, and two donor sites were randomly assigned to experience either natural healing or healing-assisted with PRP. The outcome was assessed based on the comparison of the extent of wound closure, Manchester scale, Landry healing scale, visual analog scale, and tissue thickness between the study groups at different time intervals. STATISTICAL ANALYSIS USED: Repeated measurements of analysis of variance and paired t-test were used. Statistical significance was P ≤ 0.05. RESULTS: Significant differences between the study groups and also across different time intervals were seen in all parameters except for the changes in tissue thickness. CONCLUSION: PRP accelerates the healing process of wounds and reduces the healing time.

Genomic Profiling of Chronic Myelogenous Leukemia: Basic and Clinical Approach.

Chronic myeloid leukemia (CML) is a hematological stem cell cancer driven by BCR-ABL1 fusion protein. We review the previous and recent evidence on the significance of CML in diagnostic and clinic management. The technical monitoring of BCR-ABL1 with quantitative real time-PCR has been used in assessing patient outcome. The cytogenetic mark of CML is Philadelphia chromosome, that is formed by reciprocal chromosomal translocations between human chromosome 9 and 22, t(9:22) (q34:q11). It makes a BCR-ABL1 fusion protein with an anomaly tyrosine kinase activity that promotes the characteristic proliferation of progenitor cells in CML and acute lymphoblastic lymphoma. The targeting of BCR-ABL1 fusion kinase is the first novel paradigm of molecularly targeted curing.

Effect of feed supplementation with Satureja khuzistanica essential oil on performance and physiological parameters of broilers fed on wheat- or maize-based diets.

1. The aim of this study was to evaluate various levels of Satureja khuzistanica essential oil (SKEO) on performance and physiological parameters of broilers fed on wheat- or maize-based diets. 2. Treatments consisted of two cereals (maize- or wheat-based diets) and three levels of SKEO)0, 250 and 500 mg/kg). 3. Feed intake (FI) and body weight gain (BWG) decreased in broilers fed on the wheat-based diets compared to the control diet. Dietary supplementation of 500 mg/kg SKEO increased FI and BWG. Dietary SKEO supplementation was effective in improving FI and BWG only in wheat-based diets and was effective in improving feed conversion ratio (FCR) in both diets. 4. Dietary supplementation of 500 mg/kg SKEO caused a significant decrease in plasma cholesterol and triglycerides. Also, plasma low-density lipoprotein concentration was decreased in broilers fed on 250 and 500 mg/kg of SKEO. Plasma cholesterol levels in birds fed on the wheat-based diets were lower than in those fed on maize-based diets. 5. Dietary supplementation of 500 mg/kg SKEO increased caecal population of Lactobacillus and reduced total bacterial and Escherichia coli count. Caecal population of Lactobacillus decreased in broilers fed on the wheat-based diets. 6. Digesta viscosity was increased in broilers fed on the wheat-based diets and decreased in broilers fed on 500 mg/kg SKEO compared to the control diet. Dietary SKEO supplementation was effective in reducing digesta viscosity values only in wheat-based diets. 7. Villus height of the duodenum and jejunum decreased in broilers fed on the wheat-based diets. Dietary supplementation of 500 mg/kg SKEO increased villus height and villus height-to-crypt depth ratios and decreased crypt depth of the duodenum compared to the control diet. 8. It can be concluded that dietary SKEO supplementation was effective in improving FI, BWG and FCR values in wheat-based diets.

Early versus late traumatic cataract surgery and intraocular lens implantation.

PurposeTo determine the proper time for traumatic cataract surgery after open globe injuries.SettingFarabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran.DesignRandomized clinical trial.Patients and methodsIn a randomized clinical trial, 30 eyes with traumatic cataract after open globe injury with IOL implantation underwent early (in the first week after the trauma) and 30 eyes underwent late cataract surgery (from the first to second month after the trauma). We excluded patients who were under 12-year-old. All patients were visited at 1 week, 4 weeks, 12 weeks, and 6 months after surgery. In each visit, patients were examined regarding visual acuity, intraocular pressure (IOP), anterior chamber inflammation, IOL position, and posterior synechiae. In addition, posterior segment evaluation and fundoscopy were performed. Intraoperative complication including posterior capsular rupture, anterior vitrectomy, and zonulysis as well as the site of IOL implantation were documented and post-operative complications including raised IOP, anterior chamber inflammation, visual axis opacity, posterior synechiae, subluxation of IOL, and IOL pigment deposition were listed.ResultsBest-corrected visual acuity 6 months after surgery was not different between the two groups. Also in early cataract surgery group, the rate of posterior capsular rupture was not significantly higher than the late surgery group (P=0.069). On the other hand, zonulysis was significantly higher in the late procedure group (P=0.039). Other complications including anterior vitrectomy, raised IOP, anterior chamber inflammation, visual axis opacity, posterior synechiae, subluxation of IOL, and IOL pigment deposition were not different in the two groups.ConclusionsEarly and late traumatic cataract surgery and IOL implantation after open globe injuries, have no significant difference regarding the post-surgical BCVA and prominent intraoperative and post-operative complications.

A histologic, histomorphometric, and radiographic comparison between two complexes of CenoBoen/CenoMembrane and Bio-Oss/Bio-Gide in lateral ridge augmentation: A clinical trial.

BACKGROUND: Several grafting materials have been used for alveolar ridge augmentation. The literature lacks researches to compare CenoBone to other grafting materials. The aim of this study was to compare CenoBone/CenoMembrane complex to Bio-Oss/Bio-Gide complex in lateral alveolar bone augmentation in terms of radiographic, histologic, and histomorphometric parameters. MATERIALS AND METHODS: In this randomized controlled trial, ten patients who needed lateral ridge augmentation were selected and augmentations were done using either of CenoBone/CenoMembrane or Bio-Oss/Bio-Gide complexes. In the re-entry surgery in 6 months following augmentation, core biopsies were taken and clinical, radiographic, histologic, and histomorphometric evaluations were performed. RESULTS: No statistically significant difference was seen between groups except for the number of blood vessels and percentage of residual graft materials. CONCLUSION: CenoBone seems to present a comparable lateral ridge augmentation to Bio-Oss in.

Detection of Superior Markers for Polymerase Chain Reaction Diagnosis of Breast Cancer Micrometastasis in Sentinel Lymph Nodes.

Breast cancer is the most prevalent type of cancer among women around the world, and mortality is primarily caused by micro-metastatic disease. The complex mechanisms of breast cancer invasion and metastasis are intrinsically related to the malignant cell type so that early detection of micro-metastases can help prolongation of survival for patient. The aim of the present research work was evaluation of the expression status of mammoglobin protein as a candidate molecular marker in the negative sentinel lymph node (SLN). Fifty tumor specimens, and 50 normal adjacent breast tissue samples from the same patients were selected on the basis of having more than 10% tumor content for RNA extraction from SLNs. Tumor samples and normal adjacent breast tissue were archived in the form of frozen fresh tissue in liquid nitrogen. Real-time PCR was performed on a Bioner life express gradient thermal cycler system. Mammoglobin gene overexpression in breast cancer metastasis was investigated. Single marker results were mammaglobin 66.7% and CK19 50.0%, with 58.3% for the two in combination. Due to improved outcome with at least 3 genes (83.3%), it seems, triple marker evaluation will be most likely useful for detecting micro-metastases instead of studying separate genes.

Targeting of BUB1b Gene Expression in Sentinel Lymph Node Biopsies of Invasive Breast Cancer in Iranian Female Patients.

Detection of micrometastasis in sentinel lymph nodes (SLNs) is a very useful tool for appropriate assessment of the clinical stage of disease in breast cancer patients. Early identification of clinically relevant disease could lead to early treatment or staging approaches for breast cancer patient. Micrometastases in SLNs of women with invasive breast cancer are of great significance in this context. In this study we examined SLN biopsies considered to have small numbers of cancerous cells by real time RT-PCR. All of the samples underwent immunohistochemical staining for cytokeratin for confirmation of the presence or absence of micrometastases. BUB1b expression assay of selected patients with and without metastasis showed overexpression in the former, but not in normal breast and lymph node tissue. Our results may be taken into account in the discussion about the merits of routine use of molecular assessment in pathogenetic studies of SLNs.

Birth outcomes in a tertiary teaching hospitals and local outposts: a novel approach to service delivery from Iran.

OBJECTIVE: The aim of this study was to compare the outcomes of childbirth care in a tertiary teaching hospital and Safe Delivery Posts (SDPs) to determine the safety of out-of-hospital care by midwives in Zahedan, Iran. STUDY DESIGN: A quasi-experimental design was applied in this study. METHODS: In this study, 2063 women who gave birth in SDPs, along with 983 women who underwent vaginal delivery in a tertiary teaching hospital, were evaluated in 2011-2012. Retrospective chart review was applied to collect data from the medical records of mothers and neonates. Only low-risk women with a singleton live birth, cephalic presentation, gestational age ≥37 weeks, spontaneous labour, and no prior history of uterine scar were recruited. RESULTS: Based on the findings, episiotomy, perineal tear, cervical laceration, postpartum haemorrhage and need for blood transfusion (or hysterectomy) were less commonly reported in the SDP group, compared to the hospital group. In the SDP group, 15 (0.73%) women were transferred to the hospital after delivery. Overall, one (0.10%) case from the hospital group and two (0.10%) cases from the SDP group were admitted to the intensive care unit. One-minute Apgar score lower than seven, resuscitation, NICU admission and neonatal death were more commonly reported in the hospital group, compared to the SDP group. Overall, hospital transfer was reported in 12 (0.58%) neonates born in SDPs. CONCLUSION: In the present study, women who gave birth in SDPs had more opportunities to experience natural birth with fewer adverse outcomes. However, considering the possibility of life-threatening complications for mothers and newborns, substantial evidence is required to improve the quality of care before implementing such novel strategies in different settings.