Proteome Profiling of Recombinant DNase Therapy in Reducing NETs and Aiding Recovery in COVID-19 Patients.

Severe coronavirus disease 2019 (COVID-19) can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hallmark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe patients with COVID-19 reveal high levels of neutrophil extracellular trap (NET) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and lead to acute respiratory distress syndrome. Recombinant human DNase (Pulmozyme; Roche) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated five patients pwith COVID-19 resenting acute symptoms with clinically approved aerosolized Pulmozyme. No adverse reactions to the drug were seen, and improved oxygen saturation and recovery in all severely ill patients with COVID-19 was observed after therapy. Immunofluorescence and proteome analysis of sputum and blood plasma samples after treatment revealed a marked reduction of NETs and a set of statistically significant proteome changes that indicate reduction of hemorrhage, plasma leakage and inflammation in the airways, and reduced systemic inflammatory state in the blood plasma of patients. Taken together, the results indicate that NETs contribute to acute respiratory failure in COVID-19 and that degrading NETs may reduce dependency on external high-flow oxygen therapy in patients. Targeting NETs using recombinant human DNase may have significant therapeutic implications in COVID-19 disease and warrants further studies.

Evaluation of micronuclei and antioxidant status in hospital radiation workers occupationally exposed to low-dose ionizing radiation.

PURPOSE: There is scientific evidence that ionizing radiation (IR) can be responsible for various health hazards that are one of the concerns in occupational exposure. This study was performed to evaluate DNA damage and antioxidant status in hospital workers who are occupationally exposed to low doses of IR. MATERIALS AND METHODS: In this study, twenty occupationally exposed to low doses of IR (CT and angiography) comprising with control groups which matched them. In order to investigate the effects of chronic irradiation of radiation workers, Micronuclei (MN) frequency and the antioxidant activity of Superoxide Dismutase (SOD), Catalase (CAT) and Total Antioxidant Capacity (TAC) were measured. Then, to check adaptation against high challenge dose, the samples (in all groups) were irradiated in vitro and MN frequency was compared. Finally, to investigated the effect of the high dose after the acute and chronic low dose of ionizing radiation, MN frequency was compared in two groups (the control group that was to in-vitro irradiated (acute low dose + high dose) and radiation workers (chronic low dose + high dose)). RESULTS: MN frequency in the occupationally exposed group (n = 30) increased significantly when compared to the control group (p-value < 0.0001). However, chronic irradiation of radiation workers could not lead to an adaptive Sresponse, while acute low-doses could produce this effect (p-value ˂ 0.05). In addition, the activity levels of antioxidant enzymes SOD, CAT, and TAC were not statistically different between the radiation workers and the control group (p-value > 0.05). CONCLUSIONS: We observed that exposure to low doses of IR leads to increased cytogenetic damage, could not cause an adaptive-response, and improve antioxidant capacity in radiation workers. Controlling healthcare workers' exposure is the first step to improving the health of hospital workers and the quality of patient care, thus decreasing human and economic costs.

DNA vaccination with a plasmid encoding LACK-TSA fusion against Leishmania major infection in BALB/c mice.

Vaccination would be the most important strategy for the prevention and elimination of leishmaniasis. The aim of the present study was to compare the immune responses induced following DNA vaccination with LACK (Leishmania analogue of the receptor kinase C), TSA (Thiol-specific-antioxidant) genes alone or LACK-TSA fusion against cutaneous leishmaniasis (CL). Cellular and humoral immune responses were evaluated before and after challenge with Leishmania major (L. major). In addition, the mean lesion size was also measured from 3th week post-infection. All immunized mice showed a partial immunity characterized by higher interferon (IFN)-γ and Immunoglobulin G (IgG2a) levels compared to control groups (p<0.05). IFN-γ/ Interleukin (IL)-4 and IgG2a/IgG1 ratios demonstrated the highest IFN-γ and IgG2a levels in the group receiving LACK-TSA fusion. Mean lesion sizes reduced significantly in all immunized mice compared with control groups at 7th week post-infection (p<0.05). In addition, there was a significant reduction in mean lesion size of LACK-TSA and TSA groups than LACK group after challenge (p<0.05). In the present study, DNA immunization promoted Th1 immune response and confirmed the previous observations on immunogenicity of LACK and TSA antigens against CL. Furthermore, this study demonstrated that a bivalent vaccine can induce stronger immune responses and protection against infectious challenge with L. major.

The phenotypic evolution of Pseudomonas aeruginosa populations changes in the presence of subinhibitory concentrations of ciprofloxacin.

Ciprofloxacin is a widely used antibiotic, in the class of quinolones, for treatment of Pseudomonas aeruginosa infections. The immediate response of P. aeruginosa to subinhibitory concentrations of ciprofloxacin has been investigated previously. However, the long-term phenotypic adaptation, which identifies the fitted phenotypes that have been selected during evolution with subinhibitory concentrations of ciprofloxacin, has not been studied. We chose an experimental evolution approach to investigate how exposure to subinhibitory concentrations of ciprofloxacin changes the evolution of P. aeruginosa populations compared to unexposed populations. Three replicate populations of P. aeruginosa PAO1 and its hypermutable mutant ΔmutS were cultured aerobically for approximately 940 generations by daily passages in LB medium with and without subinhibitory concentration of ciprofloxacin and aliquots of the bacterial populations were regularly sampled and kept at - 80 °C for further investigations. We investigate here phenotypic changes between the ancestor (50 colonies) and evolved populations (120 colonies/strain). Decreased protease activity and swimming motility, higher levels of quorum-sensing signal molecules and occurrence of mutator subpopulations were observed in the ciprofloxacin-exposed populations compared to the ancestor and control populations. Transcriptomic analysis showed downregulation of the type III secretion system in evolved populations compared to the ancestor population and upregulation of denitrification genes in ciprofloxacin-evolved populations. In conclusion, the presence of antibiotics at subinhibitory concentration in the environment affects bacterial evolution and further studies are needed to obtain insight into the dynamics of the phenotypes and the mechanisms involved.

The twin-arginine translocation system is vital for cell adhesion and uptake of iron in the cystic fibrosis pathogen Achromobacter xylosoxidans.

BACKGROUND: Achromobacter xylosoxidans is an emerging pathogen that causes airway infections in patients with cystic fibrosis. Knowledge of virulence factors and protein secretion systems in this bacterium is limited. Twin arginine translocation (Tat) is a protein secretion system that transports folded proteins across the inner cell membranes of gram-negative bacteria. Tat has been shown to be important for virulence and cellular processes in many different bacterial species. This study aimed to investigate the role of Tat in iron metabolism and host cell adhesion in A. xylosoxidans. METHODS: Putative Tat substrates in A. xylosoxidans were identified using the TatFind, TatP, and PRED-Tat prediction tools. An isogenic tatC deletion mutant (ΔtatC) was generated and phenotypically characterized. The wild-type and ΔtatC A. xylosoxidans were fractionated into cytosolic, membrane, and periplasmic fractions, and the expressed proteome of the different fractions was analyzed using liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: A total of 128 putative Tat substrates were identified in the A. xylosoxidans proteome. The ΔtatC mutant showed attenuated host cell adhesion, growth rate, and iron acquisition. Twenty predicted Tat substrates were identified as expressed proteins in the periplasmic compartment, nine of which were associated with the wild type. CONCLUSION: The data indicate that Tat secretion is important for iron acquisition and host cell adhesion in A. xylosoxidans.

Genomic and Phenotypic Evolution of Achromobacter xylosoxidans during Chronic Airway Infections of Patients with Cystic Fibrosis.

Bacterial pathogens evolve during chronic colonization of the human host by selection for pathoadaptive mutations. One of the emerging and understudied bacterial species causing chronic airway infections in patients with cystic fibrosis (CF) is Achromobacter xylosoxidans. It can establish chronic infections in patients with CF, but the genetic and phenotypic changes associated with adaptation during these infections are not completely understood. In this study, we analyzed the whole-genome sequences of 55 clinical A. xylosoxidans isolates longitudinally collected from the sputum of 6 patients with CF. Four genes encoding regulatory proteins and two intergenic regions showed convergent evolution, likely driven by positive selection for pathoadaptive mutations, across the different clones of A. xylosoxidans. Most of the evolved isolates had lower swimming motility and were resistant to multiple classes of antibiotics, while fewer of the evolved isolates had slower growth or higher biofilm production than the first isolates. Using a genome-wide association study method, we identified several putative genetic determinants of biofilm formation, motility and ß-lactam resistance in this pathogen. With respect to antibiotic resistance, we discovered that a combination of mutations in pathoadaptive genes (phoQ and bigR) and two other genes encoding regulatory proteins (spoT and cpxA) were associated with increased resistance to meropenem and ceftazidime. Altogether, our results suggest that genetic changes within regulatory loci facilitate within-host adaptation of A. xylosoxidans and the emergence of adaptive phenotypes, such as antibiotic resistance or biofilm formation. IMPORTANCE A thorough understanding of bacterial pathogen adaptation is essential for the treatment of chronic bacterial infections. One unique challenge in the analysis and interpretation of genomics data is identifying the functional impact of mutations accumulated in the bacterial genome during colonization in the human host. Here, we investigated the genomic and phenotypic evolution of A. xylosoxidans in chronic airway infections of patients with CF and identified several mutations associated with the phenotypic evolution of this pathogen using genome-wide associations. Identification of phenotypes under positive selection and the associated mutations can enlighten the adaptive processes of this emerging pathogen in human infections and pave the way for novel therapeutic interventions.

Within-Host Adaptation Mediated by Intergenic Evolution in Pseudomonas aeruginosa.

Bacterial pathogens evolve during the course of infection as they adapt to the selective pressures that confront them inside the host. Identification of adaptive mutations and their contributions to pathogen fitness remains a central challenge. Although mutations can either target intergenic or coding regions in the pathogen genome, studies of host adaptation have focused predominantly on molecular evolution within coding regions, whereas the role of intergenic mutations remains unclear. Here, we address this issue and investigate the extent to which intergenic mutations contribute to the evolutionary response of a clinically important bacterial pathogen, Pseudomonas aeruginosa, to the host environment, and whether intergenic mutations have distinct roles in host adaptation. We characterize intergenic evolution in 44 clonal lineages of P. aeruginosa and identify 77 intergenic regions in which parallel evolution occurs. At the genetic level, we find that mutations in regions under selection are located primarily within regulatory elements upstream of transcriptional start sites. At the functional level, we show that some of these mutations both increase or decrease transcription of genes and are directly responsible for evolution of important pathogenic phenotypes including antibiotic sensitivity. Importantly, we find that intergenic mutations facilitate essential genes to become targets of evolution. In summary, our results highlight the evolutionary significance of intergenic mutations in creating host-adapted strains, and that intergenic and coding regions have different qualitative contributions to this process.

Peritonsillar infiltration with bupivacaine and pethidine for relief of post-tonsillectomy pain: a randomised double-blind study.

Previous studies of infiltration of local anaesthetics in children undergoing tonsillectomy resulted in conflicting results. The aim of this study was to evaluate the effect of the peritonsillar injection of bupivacaine and pethidine on postoperative pain in children undergoing snare-dissection tonsillectomy. In a double-blind study, 80 children (aged 7-15 years) were randomly divided into two groups receiving peritonsillar injection of either bupivacaine (1 mg x kg(-1)) and pethidine (1 mg x kg(-1)) in adrenaline 1:200,000 (treatment group) or an equivalent volume of saline (placebo group) pre-operatively. The time needed for first demand of analgesia and analgesic consumption to reduce the visual analogue scale (VAS) for resting throat pain to < or = 30, the VAS for pain on swallowing, drinking liquid and eating a soft diet, incidence of nausea and vomiting, and the need for rescue anti-emetics in the first 24 h after operation were compared in both groups. The combination of bupivacaine and pethidine could significantly decrease the consumption of analgesics for resting pain at 4, 6, 8, 12, and 24 h after operation but did not reduce pain on swallowing, drinking liquid and eating a soft diet. The times to demand of first dose of analgesic and to first oral intake were not significantly different. The overall satisfaction of patients in relation to relief of postoperative pain was not significantly different between the two groups. Although peritonsillar injection of pethidine and bupivacaine in children reduces the analgesic consumption, it does not affect the dynamic pain state in the first 24 h after snare-dissection tonsillectomy.

Evaluation of Radiation Exposure Pattern and Radiation Absorbed Dose Resulting from Occupational Exposure of Anesthesiologists to Ionizing Radiation.

INTRODUCTION: Little information is available concerning the radiation exposure of anesthesiologists, and no such data have previously been collected in Iran. This prospective study was performed to determine the amount of radiation exposure of anesthesiologists for the purpose of assessing whether or not dangerous levels of radiation exposures were being reached, and to identify factors that correlate with excessive risk. PARTICIPANTS AND METHODS: The radiation exposure of all anesthesiology residents and the attending of Shiraz University of Medical Sciences during a 3-month period (from June to August 2016) was measured using a film badge with monthly readings. Physicians were divided into two groups: group 1 (the ones assigned to ORs with radiation exposure), and group 2 (the ones assigned to ORs with no or minimal radiation exposure). RESULTS: A total number of 10744 procedures were performed in 3 major university hospitals including 353 cases of pediatric angiography, 251 cases of percutaneous nephrolithotomy, 43 cases of chronic pain palliation and 672 cases of orthopedic surgeries with C-arm application. In all 3 months, there were statistically significant differences in the amount of radiation exposure between the two groups. CONCLUSION: Anesthesiologists working in the cardiac catheterization laboratory, pain treatment service, orthopedic and urologic ORs are exposed to statistically significantly higher radiation levels compared to their colleagues in other ORs. The radiation exposure to anesthesiologists can rise to high levels; therefore, they should get proper teaching, shielding and periodic evaluations.

Measurement of Thyroid Dose by TLD arising from Radiotherapy of Breast Cancer Patients from Supraclavicular Field.

BACKGROUND: Breast cancer is the most frequently diagnosed cancer and the leading global cause of cancer death among women worldwide. Radiotherapy plays a significant role in treatment of breast cancer and reduces locoregional recurrence and eventually improves survival. The treatment fields applied for breast cancer treatment include: tangential, axillary, supraclavicular and internal mammary fields. OBJECTIVE: In the present study, due to the presence of sensitive organ such as thyroid inside the supraclavicular field, thyroid dose and its effective factors were investigated. MATERIALS AND METHODS: Thyroid dose of 31 female patients of breast cancer with involved supraclavicular lymph nodes which had undergone radiotherapy were measured. For each patient, three TLD-100 chips were placed on their thyroid gland surface, and thyroid doses of patients were measured. The variables of the study include shield shape, the time of patient's setup, the technologists' experience and qualification. Finally, the results were analyzed by ANOVA test using SPSS 11.5 software. RESULTS: The average age of the patients was 46±10 years. The average of thyroid dose of the patients was 140±45 mGy (ranged 288.2 and 80.8) in single fraction. There was a significant relationship between the thyroid dose and shield shape. There was also a significant relationship between the thyroid dose and the patient's setup time. CONCLUSION: Beside organ at risk such as thyroid which is in the supraclavicular field, thyroid dose possibility should be reduced. For solving this problem, an appropriate shield shape, the appropriate time of the patient's setup, etc. could be considered.

Differential regulation of basic helix-loop-helix mRNAs in the dentate gyrus following status epilepticus.

In various chemoconvulsant models of human temporal lobe epilepsy, the induction of epileptogenesis by a prolonged period of continuous seizure activity is accompanied by significant changes in hippocampal structure. These changes include an increase in neurogenesis within the proliferative subgranular zone (SGZ) of the dentate gyrus and induction of mossy fiber sprouting in mature dentate granule cells. As dentate granule cell neurogenesis and axon outgrowth are also hallmarks of hippocampal development, we hypothesized that molecules involved in normal development may also play a role in similar changes associated with epileptogenesis. To begin to test this hypothesis, we have analyzed the expression patterns of multiple members of the basic helix-loop-helix (bHLH) family of transcription factors in both normal and epileptic adult rats. bHLH protein expression has been found recently in dentate granule cells at specific developmental stages, and analysis of developmental models suggests specific neural differentiation functions for these molecules. We show that mRNA expression of all seven bHLH family members examined in this study, as well as the divergent homeobox protein Prox1, is present in the adult. Patterns of expression varied considerably between family members, ranging from the limited expression of Mash1 in the neurogenic SGZ of the dentate gyrus to the scattered, widespread profile of Hes5 throughout the dentate gyrus and the hippocampus proper. Moreover, these varied profiles of expression were differentially regulated following status epilepticus, with some increasing (Mash1, Id2), some falling (Hes5, Prox1), and others remaining mostly unchanged (NeuroD/BETA2, NeuroD2/NDRF, Id3, Rath2/Nex1). While the function of these molecules in the adult brain remains to be characterized, our findings support the idea that molecules controlling cell-fate decisions in the developing dentate gyrus are also operative during seizure-induced neurogenesis and plasticity.

The mechanism of action and mode of inhibition of dihydroorotate dehydrogenase. A quantum chemical study.

Semiemperical quantum chemical calculations have been applied to study the reaction mechanism and mode of inhibition of dihydroorotate dehydrogenase. The structure of substrate, intermediate, product and various inhibitors of dihydroorotate dehydrogenase were optimized using MNDO method and the geometry, heat of formation and the net atomic partial charges of optimized molecules, as well as the energy of the reaction path were calculated. This study shows that the carbanion intermediate of this reaction is rather stable (heat of formation = -134.5 kcal) and readily forms upon nucleophilic attack by groups such as hydroxyl ion. There is good correlation between electronic properties and the biological activities of various inhibitors of this enzyme and the geometry of the most active inhibitor resembles closely that of the intermediate of the reaction. Therefore, it is concluded that the enzymatic oxidation of dihydroorotate dehydrogenase proceeds via formation of an intermediate and the inhibitors bind to the active site of this enzyme in the place of this intermediate.

Comparing the attitudes of urban and rural Iranian women toward menopause.

OBJECTIVES: This study was designed to compare the attitudes of urban and rural Iranian women toward menopause. The socio-cultural context of urban Tehranian women is considerably more westernized than that of rural Semiromian women. Our original hypothesis was that urban Tehranian women would have a more negative outlook on menopause than rural women. This hypothesis was drawn from previous work implicating the youth-oriented culture of the west as responsible for the negative attitude of western women toward menopause. METHODS: A questionnaire with eight questions regarding major psychosocial issues that are of importance to menopausal women was prepared and administered to a group of 70 Tehranian and 49 Semiromian women. RESULTS: In contrast to the initial hypothesis, analysis revealed that rural Iranian women have a more negative attitude towards menopause than urban Iranian women. CONCLUSION: The more negative attitude of the rural women toward menopause in large part reflected the higher priority they placed on fertility than did their urban counterparts. Further, cross-cultural studies will illuminate exactly which social and cultural factors play a role in both the physical and psychological aspects of menopause.

Within-host evolution of Pseudomonas aeruginosa reveals adaptation toward iron acquisition from hemoglobin.

ABSTRACT Pseudomonas aeruginosa airway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist, P. aeruginosa depends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and the Pseudomonas heme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation of P. aeruginosa to the host environment. Here we investigated the within-host evolution of the transmissible P. aeruginosa DK2 lineage. We found positive selection for promoter mutations leading to increased expression of the phu system. By mimicking conditions of the CF airways in vitro, we experimentally demonstrate that increased expression of phuR confers a growth advantage in the presence of hemoglobin, thus suggesting that P. aeruginosa evolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additional P. aeruginosa lineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages, phuR promoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment of P. aeruginosa infections in CF patients. IMPORTANCE Most bacterial pathogens depend on scavenging iron within their hosts, which makes the battle for iron between pathogens and hosts a hallmark of infection. Accordingly, the ability of the opportunistic pathogen Pseudomonas aeruginosa to cause chronic infections in cystic fibrosis (CF) patients also depends on iron-scavenging systems. While the regulation and mechanisms of several such iron-scavenging systems have been well described, not much is known about how the within-host selection pressures act on the pathogens' ability to acquire iron. Here, we investigated the within-host evolution of P. aeruginosa, and we found evidence that P. aeruginosa during long-term infections evolves toward iron acquisition from hemoglobin. This adaptive strategy might be due to a selective loss of other iron-scavenging mechanisms and/or an increase in the availability of hemoglobin at the site of infection. This information is relevant to the design of novel CF therapeutics and the development of models of chronic CF infections.

Ambipolar charge transport in "traditional" organic hole transport layers.

Organic semiconductors are often labeled as electron or hole transport materials due to the primary role they perform in devices. However, despite these labels we have shown using time-of-flight that two of the traditional "hole transport materials" TPD and NPB are actually excellent electron transporters the electron transport properties of which are comparable to those for holes.

Construction, expression, and characterization of a baculovirally expressed catalytic domain of human matrix metalloproteinase-9.

We report DNA construction, baculovirus expression, and partial characterization of a minienzyme form of the human matrix metalloproteinase-9 (MMP-9). The MMP-9 minienzyme gene construct consisting of the pre, pro, and catalytic domains of the MMP-9 was introduced into Sf9 insect cells using a baculovirus expression system. The expression of the recombinant MMP-9 minienzyme was estimated to be approximately 0.8 mg/L of cell medium. The recombinant protein was purified using a single-step gelatin-Sepharose affinity column and yielded a highly stable and active minienzyme with gelatinolytic activity. Moreover, two interesting findings related to MMP-9 interactions with heparin and TIMP-1 resulted from our studies. First, the pro and catalytic domains of the human MMP-9 are not sufficient for heparin affinity. Second, in contrast to the prevailing consensus, TIMP-1 blockade of the enzymatic activity of MMP-9 does not require prior binding to the C-terminus of its MMP-9 protein substrate.