Radiation therapy with phenotypic medicine: towards N-of-1 personalization.

In current clinical practice, radiotherapy (RT) is prescribed as a pre-determined total dose divided over daily doses (fractions) given over several weeks. The treatment response is typically assessed months after the end of RT. However, the conventional one-dose-fits-all strategy may not achieve the desired outcome, owing to patient and tumor heterogeneity. Therefore, a treatment strategy that allows for RT dose personalization based on each individual response is preferred. Multiple strategies have been adopted to address this challenge. As an alternative to current known strategies, artificial intelligence (AI)-derived mechanism-independent small data phenotypic medicine (PM) platforms may be utilized for N-of-1 RT personalization. Unlike existing big data approaches, PM does not engage in model refining, training, and validation, and guides treatment by utilizing prospectively collected patient's own small datasets. With PM, clinicians may guide patients' RT dose recommendations using their responses in real-time and potentially avoid over-treatment in good responders and under-treatment in poor responders. In this paper, we discuss the potential of engaging PM to guide clinicians on upfront dose selections and ongoing adaptations during RT, as well as considerations and limitations for implementation. For practicing oncologists, clinical trialists, and researchers, PM can either be implemented as a standalone strategy or in complement with other existing RT personalizations. In addition, PM can either be used for monotherapeutic RT personalization, or in combination with other therapeutics (e.g. chemotherapy, targeted therapy). The potential of N-of-1 RT personalization with drugs will also be presented.

The Heavy Atom Structure, "cis effect" on Methyl Internal Rotation, and 14N Nuclear Quadrupole Coupling of 1-Cyanopropene from Quantum Chemical and Microwave Spectroscopic Analysis.

The microwave spectrum of 1-cyanopropene (crotonitrile) was remeasured using two pulsed molecular jet Fourier transform microwave spectrometers operating from 2.0 to 40.0 GHz. The molecule exists in two isomer forms, E and Z, with respect to the orientation between the methyl and the cyano groups. The spectrum of the Z isomer is more intense. Due to internal rotation of the methyl group, doublets containing A and E torsional species were found for all rotational transitions. Hyperfine splittings arising from the 14N nuclear quadrupole coupling were resolved. The heavy atom structure of the Z isomer was determined by observation of 13C and 15N isotopologue spectra in natural abundances. The experimental results were supported by quantum chemistry. The complex spectral patterns were analyzed and fitted globally, and the barriers to methyl internal rotation are determined to be 478.325(28) cm-1 and 674.632(76) cm-1 for the Z and E isomers, respectively. The non-bonded intramolecular electrostatic attraction between the methyl group and the 1-cyano substituent overcomes steric hindrance, leading to higher stability of the Z isomer. The consequence is a slight opening of 3.2° of the C(1)-C(2)-C(3) angle and a radical decrease of the methyl torsional barrier in the Z isomer due to steric repulsion.

Synergy between Laminin-Derived Elastin-like Polypeptides (LELPs) Optimizes Cell Spreading.

A major component of the extracellular matrix (ECM), laminins, modulates cells via diverse receptors. Their fragments have emerging utility as components of "ECM-mimetics" optimized to promote cell-based therapies. Recently, we reported that a bioactive laminin peptide known as A99 enhanced cell binding and spreading via fusion to an elastin-like polypeptide (ELP). The ELP "handle" serves as a rapid, noncovalent strategy to concentrate bioactive peptide mixtures onto a surface. We now report that this strategy can be further generalized across an expanded panel of additional laminin-derived elastin-like polypeptides (LELPs). A99 (AGTFALRGDNPQG), A2G80 (VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT), and EF1m (LQLQEGRLHFMFD) all promote cell spreading while showing morphologically distinct F-actin formation. Equimolar mixtures of A99:A2G80-LELPs have synergistic effects on adhesion and spreading. Finally, three of these ECM-mimetics promote the neurite outgrowth of PC-12 cells. The evidence presented here demonstrates the potential of ELPs to deposit ECM-mimetics with applications in regenerative medicine, cell therapy, and tissue engineering.

Outcome of Mechanical Thrombectomy for Acute Basilar Artery Occlusion in Patients with Intracranial Atherosclerotic Disease.

INTRODUCTION: Intracranial atherosclerotic disease (ICAD) has been identified as a major cause of acute basilar artery occlusion (BAO).This study compared the characteristics and treatment outcomes in acute BAO patients with and without ICAD. METHODS: A prospective cohort study was conducted at 115 People's Hospital, Ho Chi Minh city, Vietnam from August 2021 to June 2023. Patients with acute BAO who underwent endovascular treatment within 24 h from symptom onset were included (thrombectomy alone or bridging with intravenous alteplase). The baseline characteristics and outcomes were analyzed and compared between patients with and without ICAD. Good functional outcome was defined as mRS ≤3 at 90 days. RESULTS: Among the 208 patients enrolled, 112 (53.8%) patients were categorized in the ICAD group, and 96 (46.2%) in the non-ICAD group. Occlusion in the proximal segment of the basilar artery was more common in patients with ICAD (55.4% vs. 21.9%, p < 0.001), whereas the distal segment was the most common location in the non-ICAD group (58.3% vs. 10.7%, p < 0.001). Patients in the ICAD group were more likely to undergo treatment in the late window, with a higher mean onset-to-treatment time compared to the non-ICAD group (11.6 vs. 9.5 h, p = 0.01). In multivariable logistic regression analysis, distal segment BAO was negatively associated with ICAD (aOR 0.13, 95% CI: 0.05-0.32, p < 0.001), while dyslipidemia showed a positive association (aOR 2.44, 95% CI: 1.15-5.17, p = 0.02). There was a higher rate for rescue stenting in the ICAD compared to non-ICAD group (15.2% vs. 0%, p < 0.001). However, no significant differences were found between the two groups in terms of good outcome (45.5% vs. 44.8%, p = 0.91), symptomatic hemorrhage rates (4.5% vs. 8.3%, p = 0.25), and mortality (42% vs. 50%, p = 0.25). CONCLUSION: ICAD was a common etiology in patients with BAO. The location segment of BAO and dyslipidemia were associated with ICAD in patients with BAO. There was no difference in 90-day outcomes between BAO patients with and without ICAD undergoing endovascular therapy.

Comprehensive genome­wide analysis of the chicken heat shock protein family: identification, genomic organization, and expression profiles in indigenous chicken with highly pathogenic avian influenza infection.

BACKGROUND: Heat shock proteins (HSPs) function as molecular chaperones with critical roles in chicken embryogenesis, immune response to infectious diseases, and response to various environmental stresses. However, little is known on HSP genes in chicken. In this study, to understand the roles of chicken HSPs, we performed genome-wide identification, expression, and functional analyses of the HSP family genes in chicken. RESULTS: A total of 76 HSP genes were identified in the chicken genome, which were further classified into eight distinct groups (I-VIII) based on phylogenetic tree analysis. The gene-structure analysis revealed that the members of each clade had the same or similar exon-intron structures. Chromosome mapping suggested that HSP genes were widely dispersed across the chicken genome, except in chromosomes 16, 18, 22, 25, 26, and 28-32, which lacked chicken HSP genes. On the other hand, the interactions among chicken HSPs were limited, indicating that the remaining functions of HSPs could be investigated in chicken. Moreover, KEGG pathway analysis showed that the HSP gene family was involved in the regulation of heat stress, apoptotic, intracellular signaling, and immune response pathways. Finally, RNA sequencing data revealed that, of the 76 chicken HSP genes, 46 were differentially expressed at 21 different growth stages in chicken embryos, and 72 were differentially expressed on post-infection day 3 in two indigenous Ri chicken lines infected with highly pathogenic avian influenza. CONCLUSIONS: This study provides significant insights into the potential functions of HSPs in chicken, including the regulation of apoptosis, heat stress, chaperone activity, intracellular signaling, and immune response to infectious diseases.

Tracheal reconstruction surgery for congenital tracheal stenosis.

PURPOSE: Congenital tracheal stenosis is a rare but dangerous disease. Reconstructive tracheal surgery is a life-saving treatment but also a challenging procedure. This study aims to evaluate the outcomes of tracheal reconstruction surgery. METHODS: A prospective cohort study was conducted with all the records of congenital tracheal stenosis which had been managed by tracheal reconstruction surgery at Children's Hospital 2 Ho Chi Minh City from August 2013 to August 2022. RESULTS: Sixty-seven cases, who underwent slide tracheoplasty, were included in our study. Mean age was 7.6 months (25 days - 8 years). Common congenital-associated lesion was left pulmonary artery sling, accounting for 65.7% of cases. Bronchial stenosis was found in 22.4% patients. Emergency surgery was performed in eight cases. The survival rate in this review was 86.6%. Nine patients died in which four of nine cases (44.4%) were emergency surgery. The recurrent stenosis rate was 8.9%, only two cases needed reoperation in which one died and one recovered uneventfully. The outcomes of surgery were good in 53 cases (79.1%). CONCLUSION: Tracheal reconstruction surgery with slide tracheoplasty technique is safe and versatile technique which is feasible in every case of congenital tracheal stenosis. Mortality was associated with severe cases which required emergency surgery.

Optimizing Meropenem in Highly Resistant Klebsiella pneumoniae Environments: Population Pharmacokinetics and Dosing Simulations in Critically Ill Patients.

Critically ill patients are characterized by substantial pathophysiological changes that alter the pharmacokinetics (PK) of hydrophilic antibiotics, including carbapenems. Meropenem is a key antibiotic for multidrug-resistant Gram-negative bacilli, and such pathophysiological alterations can worsen treatment outcomes. This study aimed to determine the population PK of meropenem and to propose optimized dosing regimens for the treatment of multidrug-resistant Klebsiella pneumoniae in critically ill patients. Two plasma samples were collected from eligible patients over a dosing interval. Nonparametric population PK modeling was performed using Pmetrics. Monte Carlo simulations were applied to different dosing regimens to determine the probability of target attainment and the cumulative fraction of response, taking into account the local MIC distribution for K. pneumoniae. The targets of 40% and 100% for the fraction of time that free drug concentrations remained above the MIC (ƒT>MIC) were tested, as suggested for critically ill patients. A one-compartment PK model using data from 27 patients showed high interindividual variability. Significant PK covariates were the 8-h creatinine clearance for meropenem and the presence of an indwelling catheter for pleural, abdominal, or cerebrospinal fluid drainage for the meropenem volume of distribution. The target 100% ƒT>MIC for K. pneumoniae, with a MIC of ≤2 mg/liter, could be attained by the use of a continuous infusion of 2.0 g/day. Meropenem therapy in critically ill patients could be optimized for K. pneumoniae isolates with an MIC of ≤2 mg/liter by using a continuous infusion in settings with more than 50% isolates have a MIC of ≥32mg/L.

Intracellular Dynamin Elastin-like Polypeptides Assemble into Rodlike, Spherical, and Reticular Dynasomes.

Dynamin (DNM) is a family of large GTPases possessing a unique mechanical ability to "pinch" off vesicles entering cells. DNM2 is the most ubiquitously expressed member of the DNM family. We developed a novel tool based on elastin-like polypeptide (ELP) technology to quickly, precisely, and reversibly modulate the structure of DNM2. ELPs are temperature-sensitive biopolymers that self-assemble into microdomains above sharp transition temperatures. When linked together, DNM2 and a temperature-sensitive ELP fusion organize into a range of distinct temperature-dependent structures above a sharp transition temperature, which were not observed with wild-type DNM2 or a temperature-insensitive ELP fusion control. The structures comprised three different morphologies, which were prevalent at different temperature ranges. The size of these structures was influenced by an inhibitor of the DNM2 GTPase activity, dynasore; furthermore, they appear to entrap co-expressed cytosolic ELPs. Having demonstrated an unexpected diversity of morphologically distinct structures, DNM2-ELP fusions may have applications in the exploration of dynamin-dependent biology.

Hydra-Elastin-like Polypeptides Increase Rapamycin Potency When Targeting Cell Surface GRP78.

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade.

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

Evaluation of extracellular matrix mimetic laminin bioactive peptide and elastin-like polypeptide.

The extracellular matrix (ECM) is comprised of a large network of proteins that are essential for tissue development and repair. A bioactive RGD-containing peptide from laminin α1 chain, A99 (AGTFALRGDNPQG), promotes strong cell attachment and has demonstrated utility in cell culture and tissue engineering. Various materials can be utilized as a scaffold for bioactive peptides; however, it may be advantageous to design materials that use bioconjugation strategies that do not affect bioactivity, generate homogenous products, and can be produced at scale. This report is the first to compare the methods for preparing chemically conjugated and recombinant A99 to elastin-like polypeptides (ELPs) as the scaffold and characterize the biological and cell attachment activity using human dermal fibroblasts (HDFs). ELPs are biocompatible protein-polymers that are also thermo-responsive. Below a lower critical solution temperature (LCST), they are highly soluble. Above the LCST, ELPs phase separate into a polymer-rich liquid, known as a coacervate. Both chemically conjugated and recombinant fusion between A99 and an ELP (A99-ELP-R) show dose-dependent cell attachment. In addition, coating above the LCST provides better cell spreading compared to coating at 4°C. ELPs provide an excellent structural framework for deposition of bioactive peptides of the ECM, and their intrinsic biophysical properties make laminin peptide-ELPs promising biomaterials for cell culture and tissue engineering.

Exosomal miRNA profiling from H5N1 avian influenza virus-infected chickens.

Exosomes are membrane vesicles containing proteins, lipids, DNA, mRNA, and micro RNA (miRNA). Exosomal miRNA from donor cells can regulate the gene expression of recipient cells. Here, Ri chickens were divided into resistant (Mx/A; BF2/B21) and susceptible (Mx/G; BF2/B13) trait by genotyping of Mx and BF2 genes. Then, Ri chickens were infected with H5N1, a highly pathogenic avian influenza virus (HPAIV). Exosomes were purified from blood serum of resistant chickens for small RNA sequencing. Sequencing data were analysed using FastQCv0.11.7, Cutadapt 1.16, miRBase v21, non-coding RNA database, RNAcentral 10.0, and miRDeep2. Differentially expressed miRNAs were determined using statistical methods, including fold-change, exactTest using edgeR, and hierarchical clustering. Target genes were predicted using miRDB. Gene ontology analysis was performed using gProfiler. Twenty miRNAs showed significantly different expression patterns between resistant control and infected chickens. Nine miRNAs were up-regulated and 11 miRNAs were down-regulated in the infected chickens compared with that in the control chickens. In target gene analysis, various immune-related genes, such as cytokines, chemokines, and signalling molecules, were detected. In particular, mitogen-activated protein kinase (MAPK) pathway molecules were highly controlled by differentially expressed miRNAs. The result of qRT-PCR for miRNAs was identical with sequencing data and miRNA expression level was higher in resistant than susceptible chickens. This study will help to better understand the host immune response, particularly exosomal miRNA expression against HPAIV H5N1 and could help to determine biomarkers for disease resistance.

Coactivation of Endogenous Wnt10b and Foxc2 by CRISPR Activation Enhances BMSC Osteogenesis and Promotes Calvarial Bone Regeneration.

CRISPR activation (CRISPRa) is a burgeoning technology for programmable gene activation, but its potential for tissue regeneration has yet to be fully explored. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into osteogenic or adipogenic pathways, which are governed by the Wnt (Wingless-related integration site) signaling cascade. To promote BMSC differentiation toward osteogenesis and improve calvarial bone healing by BMSCs, we harnessed a highly efficient hybrid baculovirus vector for gene delivery and exploited a synergistic activation mediator (SAM)-based CRISPRa system to activate Wnt10b (that triggers the canonical Wnt pathway) and forkhead c2 (Foxc2) (that elicits the noncanonical Wnt pathway) in BMSCs. We constructed a Bac-CRISPRa vector to deliver the SAM-based CRISPRa system into rat BMSCs. We showed that Bac-CRISPRa enabled CRISPRa delivery and potently activated endogenous Wnt10b and Foxc2 expression in BMSCs for >14 days. Activation of Wnt10b or Foxc2 alone was sufficient to promote osteogenesis and repress adipogenesis in vitro. Furthermore, the robust and prolonged coactivation of both Wnt10b and Foxc2 additively enhanced osteogenic differentiation while inhibiting adipogenic differentiation of BMSCs. The CRISPRa-engineered BMSCs with activated Wnt10b and Foxc2 remarkably improved the calvarial bone healing after implantation into the critical-sized calvarial defects in rats. These data implicate the potentials of CRISPRa technology for bone tissue regeneration.

Depth Completion and Super-Resolution with Arbitrary Scale Factors for Indoor Scenes.

Depth sensing has improved rapidly in recent years, which allows for structural information to be utilized in various applications, such as virtual reality, scene and object recognition, view synthesis, and 3D reconstruction. Due to the limitations of the current generation of depth sensors, the resolution of depth maps is often still much lower than the resolution of color images. This hinders applications, such as view synthesis or 3D reconstruction, from providing high-quality results. Therefore, super-resolution, which allows for the upscaling of depth maps while still retaining sharpness, has recently drawn much attention in the deep learning community. However, state-of-the-art deep learning methods are typically designed and trained to handle a fixed set of integer-scale factors. Moreover, the raw depth map collected by the depth sensor usually has many depth data missing or misestimated values along the edges and corners of observed objects. In this work, we propose a novel deep learning network for both depth completion and depth super-resolution with arbitrary scale factors. The experimental results on the Middlebury stereo, NYUv2, and Matterport3D datasets demonstrate that the proposed method can outperform state-of-the-art methods.

MicroRNA gga-miR-200a-3p modulates immune response via MAPK signaling pathway in chicken afflicted with necrotic enteritis.

MicroRNAs (miRNAs) are small non-coding RNAs that contribute to host immune response as post-transcriptional regulation. The current study investigated the biological role of the chicken (Gallus gallus) microRNA-200a-3p (gga-miR-200a-3p), using 2 necrotic enteritis (NE) afflicted genetically disparate chicken lines, 6.3 and 7.2, as well as the mechanisms underlying the fundamental signaling pathways in chicken. The expression of gga-miR-200a-3p in the intestinal mucosal layer of NE-induced chickens, was found to be upregulated during NE infection in the disease-susceptible chicken line 7.2. To validate the target genes, we performed an overexpression analysis of gga-miR-200a-3p using chemically synthesized oligonucleotides identical to gga-miR-200a-3p, reporter gene analysis including luciferase reporter assay, and a dual fluorescence reporter assay in cultured HD11 chicken macrophage cell lines. Gga-miR-200a-3p was observed to be a direct transcriptional repressor of ZAK, MAP2K4, and TGFß2 that are involved in mitogen-activated protein kinase (MAPK) pathway by targeting the 3'-UTR of their transcripts. Besides, gga-miR-200a-3p may indirectly affect the expression of protein kinases including p38 and ERK1/2 at both transcriptional and translational levels, suggesting that this miRNA may function as an important regulator of the MAPK signaling pathway. Proinflammatory cytokines consisting of IL-1ß, IFN-γ, IL-12p40, IL-17A, and LITAF belonging to Th1 and Th17-type cytokines, were upregulated upon gga-miR-200a-3p overexpression. These findings have enhanced our knowledge of the immune function of gga-miR-200a-3p mediating the chicken immune response via regulation of the MAPK signaling pathway and indicate that this miRNA may serve as an important biomarker of diseases in domestic animals.

k-Oligocarrageenan Promoting Growth of Hybrid Maize: Influence of Molecular Weight.

k-Oligocarrageenan (OC) is an effective biostimulator and a protector against disease infections for plants. However, the effect of OC molecular weight (MW) on plant growth is not fully understood. In this work, OCs with three different MWs (42, 17 and 4 kDa) was prepared by varying the degradation reaction time using ascorbic acid as a reagent. The product structure was confirmed by Fourier-transform infrared spectroscopy (FTIR) data. The growth promotion for maize (Zea mays L.) plants was investigated by foliar spray application of the prepared OCs. Field trials were carried out in two years, 2018 and 2019. The results showed that among treatments, OC with 4 kDa exhibited the best performance in both crop growth and grain yield parameters which indicated increases compared to the control in plant height (6.9-19.9%), length of ears (12.2%), diameter of ears (9.1%), fresh grain weight (17.8%), dry grain weight (20.0%) and grain yield (21.3%). Moreover, low MW OC augmented NP uptake in the plant growth while no effect on K uptake was observed. Therefore, OC with low MWs is potentially promising to apply as a promoter to enhance yield of crops.

Salutary factors and hospital work environments: a qualitative descriptive study of nurses in Sweden.

BACKGROUND: Extensive research describes how nurses experience their work environment. The conditions are described as stressful and dissatisfying with nurses intending to leave their workplace. Knowledge about the personal perception regarding why nurses consider leaving the hospital workplace is limited. The purpose of this study was to understand why hospital nurses remain in their workplace, which facilitates their continuation in the profession. OBJECTIVE: The objective was to explore and describe factors explaining why hospital nurses remain in the workplace. METHODS: This was a descriptive qualitative study with a purposive sample of hospital nurses in Sweden. The salutogenic theory was the basis for the interview guide and the semi-structured questions. Individual interviews were conducted in a hospital in western Sweden. Content analysis was performed to organize the coded data according to the sense of coherence. RESULTS: Data saturation was achieved with 12 interviews. Within the three themes of coherence (comprehensibility, manageability, and meaningfulness), ten subthemes were categorized from the data as follows: job satisfaction and fun at work, acknowledgement and productivity, togetherness and team security, manageable workload, variable work and challenging situations, workplace and personal space balance, collaboration and supportive leadership, valued role and good work, commitment and involvement, and pride in the professional role. CONCLUSIONS: The main findings of this study have shown the critical importance of being in a meaningful, comprehensible and manageable work context that supports nurses in maintaining their professional identity.

An improvement of real-time polymerase chain reaction system based on probe modification is required for accurate detection of African swine fever virus in clinical samples in Vietnam.

OBJECTIVE: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam. METHODS: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE. RESULTS: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78). CONCLUSION: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

Automatic Multi-Camera Extrinsic Parameter Calibration Based on Pedestrian Torsors.

Extrinsic camera calibration is essential for any computer vision task in a camera network. Typically, researchers place a calibration object in the scene to calibrate all the cameras in a camera network. However, when installing cameras in the field, this approach can be costly and impractical, especially when recalibration is needed. This paper proposes a novel, accurate and fully automatic extrinsic calibration framework for camera networks with partially overlapping views. The proposed method considers the pedestrians in the observed scene as the calibration objects and analyzes the pedestrian tracks to obtain extrinsic parameters. Compared to the state of the art, the new method is fully automatic and robust in various environments. Our method detect human poses in the camera images and then models walking persons as vertical sticks. We apply a brute-force method to determines the correspondence between persons in multiple camera images. This information along with 3D estimated locations of the top and the bottom of the pedestrians are then used to compute the extrinsic calibration matrices. We also propose a novel method to calibrate the camera network by only using the top and centerline of the person when the bottom of the person is not available in heavily occluded scenes. We verified the robustness of the method in different camera setups and for both single and multiple walking people. The results show that the triangulation error of a few centimeters can be obtained. Typically, it requires less than one minute of observing the walking people to reach this accuracy in controlled environments. It also just takes a few minutes to collect enough data for the calibration in uncontrolled environments. Our proposed method can perform well in various situations such as multi-person, occlusions, or even at real intersections on the street.

Identification of duck liver-expressed antimicrobial peptide 2 and characterization of its bactericidal activity.

OBJECTIVE: This study was conducted to identify duck liver-expressed antimicrobial peptide 2 (LEAP-2) and demonstrate its antimicrobial activity against various pathogens. METHODS: Tissue samples were collected from 6 to 8-week-old Pekin ducks (Anas platyrhynchos domesticus), total RNA was extracted, and cDNA was synthesized. To confirm the duck LEAP-2 transcript expression levels, quantitative real-time polymerase chain reaction was conducted. Two kinds of peptides (a linear peptide and a disulfide-type peptide) were synthesized to compare the antimicrobial activity. Then, antimicrobial activity assay and fluorescence microscopic analysis were conducted to demonstrate duck LEAP-2 bactericidal activity. RESULTS: The duck LEAP-2 peptide sequence showed high identity with those of other avian species (>85%), as well as more than 55% of identity with mammalian sequences. LEAP-2 mRNA was highly expressed in the liver with duodenum next, and then followed by lung, spleen, bursa and jejunum and was the lowest in the muscle. Both of LEAP-2 peptides efficiently killed bacteria, although the disulfide-type LEAP-2 showed more powerful bactericidal activity. Also, gram-positive bacteria was more susceptible to duck LEAP-2 than gram-negative bacteria. Using microscopy, we confirmed that LEAP-2 peptides could kill bacteria by disrupting the bacterial cell envelope. CONCLUSION: Duck LEAP-2 showed its antimicrobial activity against both gram-positive and gram-negative bacteria. Disulfide bonds were important for the powerful killing effect by disrupting the bacterial cell envelope. Therefore, duck LEAP-2 can be used for effective antibiotics alternatives.