Effects of hybrid post-treatments on fatigue behaviour of notched LPBF AlSi10Mg: Experimental and deep learning approaches

Laser powder bed fusion (LPBF) as one of the widely used technologies of additive manufacturing (AM), has a high capability to produce complex geometries such as notched parts in a layer-by-layer manner. LPBF parts in their as built state have inhomogeneous and anisotropic microstructure and poor surface quality. Post-treatments can play a key role in modulating these imperfections. In this study, the effects of four different post-treatments including heat treatment, shot peening and electro-chemical polishing as well as their combination as hybrid treatment were investigated on microstructure, surface and mechanical properties and finally fatigue behaviour of the LPBF V-notched AlSi10Mg samples. Afterward, a deep learning based approach was employed for modelling the fatigue behaviour via artificial neural network. Surface roughness, surface modification factor, hardness, residual stress and porosities were considered as inputs and fatigue life was considered as the output. Model function of the network was generated and the relevant parametric and sensitivity analyses were performed. The results indicated the importance of surface related properties and the notable effect of the surface post-treatments in enhancing the fatigue performance of the LPBF material

Antibacterial efficacy of lytic bacteriophages against antibiotic-resistant Klebsiella species

Bacterial resistance to antibiotics is a leading and highly prevalent problem in the treatment of infectious diseases. Bacteriophages (phages) appear to be effective and safe alternatives for the treatment of resistant infections because of their specificity for bacterial species and lack of infectivity in eukaryotic cells. The present study aimed to isolate bacteriophages against Klebsiella spp. and evaluate their efficacy against antibiotic-resistant species. Seventy-two antibiotic-resistant Klebsiella spp. were isolated from samples of patients who referred to the Ghaem Hospital (Mashhad, Iran). Lytic bacteriophages against Klebsiella spp. were isolated from wastewater of the septic tank of the same hospital. Bactericidal activity of phages against resistant Klebsiella spp. was tested in both liquid (tube method; after 1 and 24 h of incubation) and solid (double-layer agar plate method; after 24 h of incubation) phases. In each method, three different concentrations of bacteriophages (low: <10 4 PFU/mL, medium: 10 4 -10 7 PFU/mL, and high: >10 7 PFU/mL) were used. Bacteriophages showed promising bactericidal activity at all assessed concentrations, regardless of the test method and duration of incubation. Overall, bactericidal effects were augmented at higher concentrations. In the tube method, higher activity was observed after 24 h of incubation compared to the 1-h incubation. The bactericidal effects were also higher in the tube method compared to the double-layer agar plate method after 24 h of incubation. The findings of the present study suggest that bacteriophages possess effective bactericidal activity against resistant Klebsiella spp. These bactericidal activities are influenced by phage concentration, duration of incubation, and test method. KEYWORDS: bacteriophage, Klebsiella, antibiotic resistance Karamoddini et al.: Bacteriophages Against Resistant Klebsiella Species TheScientificWorldJOURNAL (2011) 11, 1332-1340 1333 INTRODUCTION Bacteriophages (also called phages) are reported to be the most abundant organisms on earth Based on the replication type, phages are classified as either lytic or lysogenic. A lytic phage replicates in the bacterial host and destroys its host in a process, but a lysogenic phage inserts itself into the genome of its bacterial host and establishes a stable position in the infected bacterium After discovery, phages were the target of multiple research for the treatment of bacterial diseases, such as dysentery In spite of the great progress that has been made in the field of antimicrobial therapy, the appearance and spread of drug-resistant bacteria has caused a serious challenge in recent decades. As an example, the prevalence of resistant nosocomial infections is increasing at an alarming rate and their elimination is very difficult. This could be secondary to the wide use of antibiotics, as well as application of therapeutic measures that weaken the immune system and make subjects more susceptible to nosocomial infections. Phage therapy could be an effective alternative approach for the control of these infections, as several studies have shown their efficacy against both Gram-positive and Gram-negative bacteria The purpose of the present study was to isolate and enrich lytic bacteriophages against Klebsiella spp. and evaluate their antibacterial efficacy against antibiotic-resistant species. The impact of phage concentration, incubation duration, and method of culture (tube vs. plate) on the bactericidal effect was also investigated. MATERIALS AND METHODS Isolation of Klebsiella spp. Different samples, mainly from urine, vaginal smears, blood, wounds and their secretions, and burn lesions, were collected from patients referring to the Ghaem Hospital (Mashhad, Iran) during a course of about 1.5 years between November 2001 and March 2003. Samples were cultured on general (simple blood agar; supporting the growth of most microorganisms) as well as specific (MacConkey agar, desoxycholate agar, or eosin methylene blue agar; supporting the growth of Gram-negative bacteria) culture media. Culture media plates were incubated at 37°C for 24 h. To confirm the isolation of Klebsiella spp., Gram staining and multiple biochemical tests were performed, including glucose and lactose fermentation (Kligler iron agar medium), citrate utilization (Simmons citrate agar medium), urea (urea agar medium), hydrogen sulfide production, indole formation and motility (sulfide-indole-motility [SIM] agar medium; Kligler iron agar medium), and malonate utilization (malonate agar medium) tests. Determination of Klebsiella spp. Sensitivity to Antibiotics Mueller-Hinton agar medium was used to culture the appropriate bacteria. Colonies were first suspended in 5 mL of tripticase soy broth and kept at 37°C for several hours until the turbidity of the suspension changed, similar to that of barium sulfate solution in the 0.5 McFarland standard tube (the standard tube was shaken vigorously before usage). A sterile swab was stirred in the above suspension and the sample was cultured on Mueller-Hinton agar medium. Antibiotic disks were placed at a 15-mm distance from the Karamoddini et al.: Bacteriophages Against Resistant Klebsiella Species TheScientificWorldJOURNAL (2011) 11, 1332-1340 1334 edge of the plate. Different disks were 24 mm from the center of each nearest disk. Following a 24-h incubation at 37°C, the growth inhibition zone was measured and compared with tables provided by the National Committee for Clinical Laboratory Standards (NCCLS). The results of sensitivity were reported as sensitive, resistant, or intermediate. Antibiotics that were evaluated included ampicillin, amoxicillin, amikacin, cephalexin, chloramphenicol, nitrofurantoin (for urine samples), gentamicin, kanamycin, nalidixic acid (for urine samples), rifampin, streptomycin, tetracycline, doxycycline, tobramycin, and sulfamethoxazole. Smooth agar containing glycerin was used to keep resistant Klebsiella colonies at -20°C as follows: four to five colonies were transferred to 20 mL of triple soy broth. After 4 h of incubation at 37°C, the tube containing tryptone soy broth was centrifuged at 2500 rpm. Then, 0.5 mL of the above-cultured bacteria was transferred to a Pyrex® test tube containing 3 mL of 3% Mueller-Hinton. Test tubes were incubated at 37°C for 4-6 h in order to accelerate bacterial growth. Following that, 0.5 mL of sterile glycerin was added to test tubes and tubes were transferred to -20°C. Isolation, Enrichment, Titration, and Bacteriophages Bacteriophages utilized in this study were isolated from wastewater of the septic tank in Ghaem Hospital that had been filter sterilized. To the aforementioned wastewater (45 mL), concentrated nutrient broth medium (5 mL) and 4-h antibiotic-resistant Klebsiella culture (5 mL) were added. Also added was 1% (v/w) MgSo 4 to provide optimum attachment of bacteriophage to bacteria. The mixture was then gently shaken and kept at 37°C for 24 h. Afterwards, chloroform was added (3 mL) and the mixture was shaken for 15 min. After being kept at room temperature for 2 h, the mixture was centrifuged (30 min, 3500 rpm) and the supernatant carefully isolated. For phage enrichment, the obtained supernatant was mixed with nutrient broth (10 mL) and 4-h Klebsiella culture (2 mL). The mixture was then processed as described above. Phage suspension was maintained in the nutrient broth at 4°C in a dark place using sterile and sealed glass containers. For the titration of phages, enriched samples were diluted by 10X in tubes containing 9 mL of tryptone broth. Then, 100 µL of each diluted sample was transferred to tubes containing 3 mL of soft agar. Afterwards, 4-h Klebsiella culture (1 mL) was added to each tube. Tubes were then shaken and their contents rapidly transferred to plates containing tryptone agar medium. The plates were incubated at 37°C for 24 h. Plates containing 30-300 plaques were used to calculate the number of phages in the primary solution using the following formula: Number of phages = Number of plaques × dilution titer × volume of media Evaluation of Antibacterial Activity The antibacterial effects of phages against antibiotic-resistant Klebsiella spp. were tested by the tube method and the double-layer agar plate method at two time points: after 1 h (for the tube method) and 24 h (for both tube and plate methods) of incubation at 37°C. In each method, three different concentrations of phages were tested: low (<10 4 PFU/mL), medium (10 4 -10 7 PFU/mL), and high (>10 7 PFU/ mL). According to the intensity of growth inhibition, the results were reported as +++ (75-100% reduction of bacteria compared to control), ++ (50-75% reduction of bacteria compared to control), + (25-50% reduction of bacteria compared to control), and -(<25% reduction of bacteria compared to control). Statistical Analysis All comparisons were performed using Fisher's exact test. A two-sided p value of <0.05 was considered to be statistically significant. Karamoddini et al.: Bacteriophages Against Resistant Klebsiella Species TheScientificWorldJOURNAL (2011) 11, 1332-1340 1335 RESULTS Out of the total samples that were collected during the course of the study (a period of approximately 1.5 years), 72 antibiotic-resistant Klebsiella spp. were isolated. Most of these species were isolated from urine, wounds, and burn lesion samples Karamoddini et al.: Bacteriophages Against Resistant Klebsiella Species TheScientificWorldJOURNAL (2011) 11, 1332-1340 1336 In the tube method, different concentrations (low, medium, and high) of phages were evaluated for their inhibitory effect against the growth of isolated, resistant Klebsiella spp. after 1 and 24 h of incubation at 37°C. The results indicated that in both time points, all three assessed concentrations had antibacterial effects without even one strain being unaffected by phage treatment. There was a marked increase in the antibacterial effects after 24 h compared to 1 h of incubation, and this was observed for all three assessed phage concentrations. There was also a positive association between phage concentration and observed antibacterial effects at both assessed time points. This effect of concentration was found to be of high statistical significance when comparing the antibacterial effects of low concentration to those of medium (p < 0.001) and high (p < 0.001) concentrations. However, while there was a significant concentration effect at the 1-h incubation time point between medium and high phage concentrations (p < 0.001), no significant difference was observed after 24 h of incubation (p > 0.05) ( DISCUSSION The most obvious result to emerge from the present study was the promising antibacterial effects of phages against resistant Klebsiella spp. at all assessed (low, medium, and high) concentrations. The results also indicated that bactericidal effects of phages are augmented with increasing concentration and time of incubation. In addition, the double-layer agar plate method was associated with higher bactericidal effects compared to the tube method. Bactericidal effects of phages at low concentrations are due to their self-replication property. At low concentrations, the number of phages is exponentially increased in the presence of bacterial host 1337 Phages possess some unique properties that make them promising candidates for the treatment of bacterial infections. First, they need to bind to specific surface receptors in order to enter the bacteria and exert their effects. Hence, their bactericidal effects would be specific. Second, since eukaryotic cells lack phage receptors, phage preparations appear be harmless to human, animal, and plant cells Several reports have demonstrated the efficacy of phages in the treatment of infectious diseases caused by Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Vibrio vulnificus, and Salmonella spp., and also Gram-positive bacteria, such as Enterococcus faecium and Staphylococcus aureus 1338 In spite of the positive findings on the therapeutic efficacy of phages, this strategy has not been introduced into routine clinical practice for the treatment of bacterial infections. This stems from several reasons, the most important of which are the advent and widespread use of antibiotics in the Western world as well as the inconsistency and unsuccessful results of early trials. The main reasons for the inconsistent findings of the early trials are (1) inadequate scientific methodology that was used; (2) not heeding the prerequisites for phage therapy, such as lack of complete knowledge on phage biology, including lysogeny phenomenon (which might have led to the employment of a wrong phage); (3) lack of placebo control and robust trial design; (4) not identifying pure phage strains; (5) not meeting safety requirements for phage preparations, such as endotoxin removal; (6) not confirming adequate phage viability in the employed preparations; and (7) rapid clearance of phages from the body. The modern generation of phage research has attempted to overcome these shortcomings and promising results have been obtained. However, there is still much work to be done in order to extrapolate positive in vitro findings into more complicated in vivo experiments In recent years, there have been relatively few studies on the efficacy of phage therapy against Klebsiella infection, particularly resistant Klebsiella spp. The promising results of this investigation add to the existing body of literature about the potential efficacy of phage therapy. As Klebsiella spp. are among the most important causes of noscomial infections As a limitation of the current study, it must be mentioned that the 24-h bacterial cultures were not tested for bacteriophage resistance. Furthermore, it would be helpful to evaluate the bactericidal efficacy of phage preparations in more detailed time points. To sum, the results of this research support the idea that phages are effective bactericidal agents that could serve as potential alternatives for antibiotics in the treatment of resistant bacterial infections. In addition, the present findings provide evidence with respect to the impact of concentration, incubation duration, and method of culture on the bactericidal effects of phages. ACKNOWLEDGMENT

A suggested new bacteriophage genus: “Viunalikevirus”

We suggest a bacteriophage genus, “Viunalikevirus”, as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ΦSH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00705-012-1360-5) contains supplementary material, which is available to authorized users

Coxsackie-adenovirus receptor expression is enhanced in pancreas from patients with type 1 diabetes

Objectives: One of the theories connecting enterovirus (EV) infection of human islets with type 1 diabetes (T1D) is the development of a fertile field in the islets. This implies induction of appropriate proteins for the viral replication such as the coxsackie–adenovirus receptor (CAR). The aim of this study was to investigate to what extent CAR is expressed in human islets of Langerhans, and what conditions that would change the expression. Design: Immunohistochemistry for CAR was performed on paraffin-embedded pancreatic tissue from patients with T1D (n=9 recent onset T1D, n=4 long-standing T1D), islet autoantibody-positive individuals (n=14) and non-diabetic controls (n=24) individuals. The expression of CAR was also examined by reverse transcription PCR on microdissected islets (n=5), exocrine tissue (n=5) and on explanted islets infected with EV or exposed to chemokines produced by EV-infected islet cells. Results: An increased frequency of patients with T1D and autoantibody-positive individuals expressed CAR in the pancreas (p<0.039). CAR staining was detected more frequently in pancreatic islets from patients with T1D and autoantibody-positive subjects (15/27) compared with (6/24) non-diabetic controls (p<0.033). Also in explanted islets cultured in UV-treated culture medium from coxsackievirus B (CBV)-1-infected islets, the expression of the CAR gene was increased compared with controls. Laser microdissection of pancreatic tissue revealed that CAR expression was 10-fold higher in endocrine compared with exocrine cells of the pancreas. CAR was also expressed in explanted islets and the expression level decreased with time in culture. CBV-1 infection of explanted islets clearly decreased the expression of CAR (p<0.05). In contrast, infection with echovirus 6 did not affect the expression of CAR. Conclusions: CAR is expressed in pancreatic islets of patients with T1D and the expression level of CAR is increased in explanted islets exposed to proinflammatory cytokines/chemokines produced by infected islets. T1D is associated with increased levels of certain chemokines/cytokines in the islets and this might be the mechanism behind the increased expression of CAR in TID islets

Snazer: the simulations and networks analyzer

<p>Abstract</p> <p>Background</p> <p>Networks are widely recognized as key determinants of structure and function in systems that span the biological, physical, and social sciences. They are static pictures of the interactions among the components of complex systems. Often, much effort is required to identify networks as part of particular patterns as well as to visualize and interpret them.</p> <p>From a pure dynamical perspective, simulation represents a relevant <it>way</it>-<it>out</it>. Many simulator tools capitalized on the "noisy" behavior of some systems and used formal models to represent cellular activities as temporal trajectories. Statistical methods have been applied to a fairly large number of replicated trajectories in order to infer knowledge.</p> <p>A tool which both graphically manipulates reactive models and deals with sets of simulation time-course data by aggregation, interpretation and statistical analysis is missing and could add value to simulators.</p> <p>Results</p> <p>We designed and implemented <it>Snazer</it>, the simulations and networks analyzer. Its goal is to aid the processes of visualizing and manipulating reactive models, as well as to share and interpret time-course data produced by stochastic simulators or by any other means.</p> <p>Conclusions</p> <p><it>Snazer </it>is a solid prototype that integrates biological network and simulation time-course data analysis techniques.</p

In vivo expression of the HBZ gene of HTLV-1 correlates with proviral load, inflammatory markers and disease severity in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP)

<p>Abstract</p> <p>Background</p> <p>Recently, human T-cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ), encoded from a minus strand mRNA was discovered and was suggested to play an important role in adult T cell leukemia (ATL) development. However, there have been no reports on the role of HBZ in patients with HTLV-1 associated inflammatory diseases.</p> <p>Results</p> <p>We quantified the HBZ and tax mRNA expression levels in peripheral blood from 56 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, 10 ATL patients, 38 healthy asymptomatic carriers (HCs) and 20 normal uninfected controls, as well as human leukemic T-cell lines and HTLV-1-infected T-cell lines, and the data were correlated with clinical parameters. The spliced HBZ gene was transcribed in all HTLV-1-infected individuals examined, whereas tax mRNA was not transcribed in significant numbers of subjects in the same groups. Although the amount of HBZ mRNA expression was highest in ATL, medium in HAM/TSP, and lowest in HCs, with statistical significance, neither tax nor the HBZ mRNA expression per HTLV-1-infected cell differed significantly between each clinical group. The HTLV-1 HBZ, but not tax mRNA load, positively correlated with disease severity and with neopterin concentration in the cerebrospinal fluid of HAM/TSP patients. Furthermore, HBZ mRNA expression per HTLV-1-infected cell was decreased after successful immunomodulatory treatment for HAM/TSP.</p> <p>Conclusion</p> <p>These findings suggest that <it>in vivo </it>expression of HBZ plays a role in HAM/TSP pathogenesis.</p

Computational Analysis of the Spatiotemporal Coordination of Polarized PI3K and Rac1 Activities in Micro-Patterned Live Cells

Polarized molecular activities play important roles in guiding the cell toward persistent and directional migration. In this study, the polarized distributions of the activities of phosphatidylinositol 3-kinase (PI3K) and the Rac1 small GTPase were monitored using chimeric fluorescent proteins (FPs) in cells constrained on micro-patterned strips, with one end connecting to a neighboring cell (junction end) and the other end free of cell-cell contact (free end). The recorded spatiotemporal dynamics of the fluorescent intensity from different cells was scaled into a uniform coordinate system and applied to compute the molecular activity landscapes in space and time. The results revealed different polarization patterns of PI3K and Rac1 activity induced by the growth factor stimulation. The maximal intensity of different FPs, and the edge position and velocity at the free end were further quantified to analyze their correlation and decipher the underlying signaling sequence. The results suggest that the initiation of the edge extension occurred before the activation of PI3K, which led to a stable extension of the free end followed by the Rac1 activation. Therefore, the results support a concerted coordination of sequential signaling events and edge dynamics, underscoring the important roles played by PI3K activity at the free end in regulating the stable lamellipodia extension and cell migration. Meanwhile, the quantification methods and accompanying software developed can provide a convenient and powerful computational analysis platform for the study of spatiotemporal molecular distribution and hierarchy in live cells based on fluorescence images

HTLV-1 Evades Type I Interferon Antiviral Signaling by Inducing the Suppressor of Cytokine Signaling 1 (SOCS1)

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of Adult T cell Leukemia (ATL) and the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the majority of HTLV-1–infected individuals remain asymptomatic carriers (AC) during their lifetime, 2–5% will develop either ATL or HAM/TSP, but never both. To better understand the gene expression changes in HTLV-1-associated diseases, we examined the mRNA profiles of CD4+ T cells isolated from 7 ATL, 12 HAM/TSP, 11 AC and 8 non-infected controls. Using genomic approaches followed by bioinformatic analysis, we identified gene expression pattern characteristic of HTLV-1 infected individuals and particular disease states. Of particular interest, the suppressor of cytokine signaling 1—SOCS1—was upregulated in HAM/TSP and AC patients but not in ATL. Moreover, SOCS1 was positively correlated with the expression of HTLV-1 mRNA in HAM/TSP patient samples. In primary PBMCs transfected with a HTLV-1 proviral clone and in HTLV-1-transformed MT-2 cells, HTLV-1 replication correlated with induction of SOCS1 and inhibition of IFN-α/β and IFN-stimulated gene expression. Targeting SOCS1 with siRNA restored type I IFN production and reduced HTLV-1 replication in MT-2 cells. Conversely, exogenous expression of SOCS1 resulted in enhanced HTLV-1 mRNA synthesis. In addition to inhibiting signaling downstream of the IFN receptor, SOCS1 inhibited IFN-β production by targeting IRF3 for ubiquitination and proteasomal degradation. These observations identify a novel SOCS1 driven mechanism of evasion of the type I IFN antiviral response against HTLV-1

G-quadruplex structures mark human regulatory chromatin

G-quadruplex (G4) structural motifs have been linked to transcription, replication and genome instability and are implicated in cancer and other diseases. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context. Herein we address this through the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find ∼10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5' UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as $\textit{MYC}$. Strikingly, $\textit{de novo}$ and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor-induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.European Molecular Biology Organization (EMBO Long-Term Fellowship), University of Cambridge, Cancer Research UK (Grant ID: C14303/A17197), Wellcome Trust (Grant ID: 099232/z/12/z