Weight Bands Research Articles

Pharmacokinetics, optimal dosing, and safety of linezolid in children with multidrug-resistant tuberculosis: Combined data from two prospective observational studies.

BackgroundLinezolid is increasingly important for multidrug-resistant tuberculosis (MDR-TB) treatment. However, among children with MDR-TB, there are no linezolid pharmacokinetic data, and its adverse effects have not yet been prospectively described. We characterised the pharmacokinetics, safety, and optimal dose of linezolid in children treated for MDR-TB.Methods and findingsChildren routinely treated for MDR-TB in 2 observational studies (2011–2015, 2016–2018) conducted at a single site in Cape Town, South Africa, underwent intensive pharmacokinetic sampling after either a single dose or multiple doses of linezolid (at steady state). Linezolid pharmacokinetic parameters, and their relationships with covariates of interest, were described using nonlinear mixed-effects modelling. Children receiving long-term linezolid as a component of their routine treatment had regular clinical and laboratory monitoring. Adverse events were assessed for severity and attribution to linezolid. The final population pharmacokinetic model was used to derive optimal weight-banded doses resulting in exposures in children approximating those in adults receiving once-daily linezolid 600 mg. Forty-eight children were included (mean age 5.9 years; range 0.6 to 15.3); 31 received a single dose of linezolid, and 17 received multiple doses. The final pharmacokinetic model consisted of a one-compartment model characterised by clearance (CL) and volume (V) parameters that included allometric scaling to account for weight; no other evaluated covariates contributed to the model. Linezolid exposures in this population were higher compared to exposures in adults who had received a 600 mg once-daily dose. Consequently simulated, weight-banded once-daily optimal doses for children were lower than those currently used for most weight bands. Ten of 17 children who were followed long term had a linezolid-related adverse event, including 5 with a grade 3 or 4 event, all anaemia. Adverse events resulted in linezolid dose reductions in 4, temporary interruptions in 5, and permanent discontinuation in 4 children. Limitations of the study include the lack of very young children (none below 6 months of age), the limited number who were HIV infected, and the modest number of children contributing to long-term safety data.ConclusionsLinezolid-related adverse effects were frequent and occasionally severe. Careful linezolid safety monitoring is required. Compared to doses currently used in children in many settings for MDR-TB treatment, lower doses may approximate current adult target exposures, might result in fewer adverse events, and should therefore be evaluated.

Organ doses in paediatric interventional cardiology. First values for patients in Costa Rica

The aim of this paper was to present the results of organ dose for paediatric patients in respect of diagnostic and therapeutic interventional cardiology procedures grouped by age and weight in Costa Rica. The data collection period was from July 2017 to July 2018. Monte Carlo software was used to calculate organ dose. The three organs for which the highest mean dose and range dose was recorded were lungs (9.5 mGy and 0.57 mGy–355.73 mGy), oesophagus (5.0 mGy and 0.36 mGy–167.88 mGy) and breast (5.0 mGy and 0.19 mGy–52.06 mGy) independent the type of procedure. The ranges of mean organ dose values by age and weight band and divided into procedure type were 0.03 (brain, 5–9 years) to 8.70 mGy (lungs, 1–4 years) for diagnostic procedures and 0.08 (brain, <1 year and 5–9 years) to 48.46 mGy (lungs, 10–15 years) for therapeutic procedures; and 0.04 (brain, 15–29 kg) to 6.98 mGy (lungs, 15–29 kg) for diagnostic procedures and 0.02 (brain, 5–14 kg and 30.49 kg) to 112.45 mGy (lungs, 50–79 kg). The resulting set of dose values will permit to assist in assessment of the effects on organ dose when new or modified irradiation techniques are introduced or, comparisons with other imaging procedures for justification purposes. Furthermore, it can also be used for further epidemiological studies on radiation risk effects in the paediatric population.

Rhabdomyosarcoma and Wilms tumors contain a subpopulation of noggin producing, myogenic cells immunoreactive for lens beaded filament proteins.

Myo/Nog cells are identified by their expression of the skeletal muscle specific transcription factor MyoD and the bone morphogenetic protein inhibitor noggin, and binding of the G8 monoclonal antibody. Their release of noggin is critical for morphogenesis and skeletal myogenesis. In the adult, Myo/Nog cells are present in normal tissues, wounds and skin tumors. Myo/Nog cells in the lens give rise to myofibroblasts that synthesize skeletal muscle proteins. The purpose of this study was to screen human lens tissue, rhabdomyosarcoma cell lines, and tissue sections from rhabdomyosarcoma, Wilms and tumors lacking features of skeletal muscle for co-localization of antibodies to Myo/Nog cell markers and the lens beaded filament proteins filensin and CP49. Immunofluorescence localization experiments revealed that Myo/Nog cells of the lens bind antibodies to beaded filament proteins. Co-localization of antibodies to G8, noggin, filensin and CP49 was observed in most RC13 and a subpopulation of RD human rhabdomyosarcoma cell lines. Western blotting with beaded filament antibodies revealed bands of similar molecular weights in RC13 and murine lens cells. Human alveolar, embryonal, pleomorphic and spindle cell rhabdomyosarcomas and Wilms tumors contained a subpopulation of cells immunoreactive for G8, noggin, MyoD and beaded filaments. G8 was also co-localized with filensin mRNA. Staining for beaded filament proteins was not detected in G8 positive cells in leiomyosarcomas, squamous and basal cell carcinomas, syringocarciomas and malignant melanomas. Lens beaded filament proteins were thought to be present only in the lens. Myo/Nog-like cells immunoreactive for beaded filaments may be diagnostic of tumors related to the skeletal muscle lineage.

Evaluation of Genetic Diversity of Seven Species of Solanum in Nigeria Using SDS-PAGE

Seed protein profile of 12 accessions belonging to 7 species of the genus Solanum of the family solanaceae were investigated through poly acrylamide gel electrophoresis. The accessions were selected based on their overlapping morphological associations observed on the field. Occurrence of two protein bands of molecular weights 100 kda and 20 kda in all accession showed interspecific relationships and could be used as generic bands among the genus Solanum. All morphologically similar accessions manifested similarities in their protein bands except S. melongena. Dendrogram based on upgma also revealed the generic status and intraspecific relationships of the accession of S. aethiopicum and S. macrocarpon.

Isoniazid mono-resistant tuberculosis: Time to take it seriously

Background/AimsIn drug resistant tuberculosis (DRTB) suspects, rifampicin resistance has always been prioritized, hence Cartridge Based Nucleic Acid Amplification Test (CBNAAT) is recommended under Revised National Tuberculosis Control Programme (RNTCP), India. However, since it doesn't detect isoniazid resistance, rifampicin sensitive patients with unknown isoniazid status may be erroneously treated as drug sensitive TB, leading to poor treatment outcomes and emergence of multidrug resistant (MDR) TB. Hence isoniazid mono-resistance should be specifically looked for and treated as per recommendations.The objective of the present study, almost the first of its kind in India, was to evaluate the burden of isoniazid mono-resistance amongst patients diagnosed with DRTB and to study the association of different patient and disease related factors with treatment outcomes under the treatment regimen specific for isoniazid mono-resistance, started from January 1, 2017 in our state, under RNTCP. MethodsIt was a retrospective study which scrutinized medical records of 52 isoniazid mono-resistant TB patients started on treatment under RNTCP between January 1 to December 31, 2017. Necessary information on possible patient and disease related predicting factors like gender, age, type of mutation (katG/inhA), weight band (26–45 kg/46-70 kg), total serum protein/albumin levels, previous history of anti-tubercular treatment (ATT), history of smoking, HIV status, presence of diabetes mellitus (DM), presence of anemia, occurrence of adverse drug reactions (ADR) during treatment and duration of intensive phase (IP), was retrieved. These factors were analyzed for their possible association with treatment outcomes. ResultsOut of 103 DRTB patients enrolled, 50.5% (52/103) patients were diagnosed with isoniazid mono-resistance. 50/103 were MDR-TB and 1/103 were extensively-drug resistant TB (XDR-TB). Further analysis of these 52 isoniazid mono-resistant patients revealed:35 (67.3%) were males and 17 (32.7%) females. 27 (51.9%) patients were <30 years, 25 (48.1%) being ≥30 years of age. All patients were negative for HIV. 34/52 (65.4%) patients were declared cured, 15/52 were lost to follow up (LTFU) and 3/52 died (1 male, 2 females). Excluding these 3 patients who died, cure rates were significantly better in females (14/15 = 93.3%), with only 1/15 LTFU, than males (20/34 = 58.8% cure, 14/34 = 41.2% LTFU), (p = 0.019). Patients who were <30 years of age had significantly better cure rates (21/25 = 84%) with lesser LTFU's (4/25 = 16%), than those ≥30years of age (13/24 = 54.2% cure, 11/24 = 45.8% LTFU), (p = 0.032). Review of previous history of ATT revealed that 33 patients had primary isoniazid mono-resistance, 4 patients had previous history of being LTFU, 9 had recurrent TB and 3 were labeled as failure. Cure rates were significantly better in primary isoniazid mono-resistant patients (26/33 = 78.8%), than those with previous history of being LTFU(0/4), (p = 0.04).Type of mutation, weight band, total serum protein/albumin, history of smoking, presence of DM, presence of anemia, occurrence of ADR and duration of IP did not affect treatment outcomes. ConclusionIsoniazid mono-resistance formed a major chunk of DRTB, with majority of the patients detected with primary mono-resistance. Strategically framed treatment regimens for isoniazid mono-resistance under RNTCP in India are effective in a wide range of population. Still, there are high chances of LTFU/default, which needs to be addressed on priority. Male gender, age ≥30 years and being LTFU in the past are associated with poorer cure rates, hence should be paid special attention.

Effect of tyrosinase‐catalyzed crosslinking on the structure and allergenicity of turbot parvalbumin mediated by caffeic acid

Enzymatic treatment of allergenic protein can alter their functional properties under a mild reaction condition due to specificity of enzymes. Phenolic compounds act as mediators and enhance the crosslinking reactions. The study aimed to assess the changes in the structure and immunoglobulin G (IgG) binding capacity of turbot parvalbumin (PV) upon crosslinking with tyrosinase (Tyr) in the absence and presence of caffeic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the appearance of higher molecular weight bands (24, 36 kDa) in the crosslinked PV. The secondary structure of crosslinked PV became loosened and disordered. The results of intrinsic fluorescence and ultraviolet absorption spectral analyses, as well as surface hydrophobicity and free amino group analyses also revealed structural changes. As observed by western blot analysis, the intensity of the PV bands reduced upon Tyr treatment, indicating reduced binding of specific IgG to PV. Moreover, the indirect ELISA (enzyme-linked immunosorbent assay) analysis confirmed that the IgG binding ability of crosslinked PV was reduced 34.94%. Enzymatic treatment mitigated the allergenicity of fish PV, which was closely related to the alterations in the conformational structure. This treatment showed potential for developing hypoallergenic fish products under mild reaction conditions. © 2019 Society of Chemical Industry.

Aerobic Versus Strengthening Exercises on Fibromyalgia in Postmenopausal Women

AbstractBackground: Fibromyalgia (FM) is a syndrome, with unknown aetiology, characterized by chronic widespread pain and several associated non-specific symptoms such as pain, fatigue, sleep disorders, depression and anxiety. It affects approximately 2-3% of the general population with more than 90% of patients being female.Aim of Study: This study was aimed to determine which is more effective in alleviating pain and fibromyalgia symp-toms; aerobic exercises or strengthening exercises in postmen-opausal women.Material and Method: Sixty postmenopausal women with FMS will be selected according to American College of Rheumatology (ACR) criteria from the Physical Therapy Department in Naser General Hospital to share in this study, their age ranged from 50-60 years and their Body Mass Index (BMI) will not exceed 30kg/m2, the participants were assigned into two groups of equal numbers. The group (A) received aerobic exercise program, in the form of treadmill 3 times/week for 8 weeks. Each session will take about 30 minutes, while the group (B) received strengthening exercises for the four limbs and trunk using their body weight or resistance band (Thera band) 3 times/week for 8 weeks. Each session will take about 30 minutes. All subjects in both groups were assessed through the Visual Analogue Scale (VAS) for pain and the Fibromyalgia Impact Questionnaire (FIQ) for the fibromyalgia related symptoms.Results: The results of the present study revealed that there was a statistically significant improvement (p<0.05) in both pain and fibromyalgia related symptoms in group (A) than in group (B).Conclusion: It can be concluded that aerobic exercises is more effective than strengthening exercises in relieving pain and fibromyalgia related symptoms in postmenopausal women.

Biotic potential of mucus extracts of giant mudskipper Periophthalmodon schlosseri (Pallas, 1770) from Pichavaram, southeast coast of India

BackgroundThe epidermal mucus of fish contains various bioactive compounds, which play a crucial role in the animal defense mechanisms. However, the mucus from giant mudskipper is not well characterized for its biochemical and pharmacological effects. This study for the first time analyzed the mucus extracts of this species for antimicrobial (against human pathogen) and hemolytic activities. Fourier-transform infrared spectrum analysis (FT-IR) and SDS-PAGE of mucus extracts were also carried out.ResultsThe protein content in Periophthalmodon schlosseri (Gobiidae: Oxudercine) mucus extracts was 0.66 ± 0.04 mg/ml. The mucus extracts of P. schlosseri showed antibacterial activity against all eight human pathogenic bacteria and four fungal strains tested in this study. The hemolytic activities against chicken and human blood cells were observed with maximum activity for dichloromethane extracts. The FT-IR spectrum of mucus extract of P. schlosseri showed the presence of functional groups, evident protein bands of various molecular weights ranging from 14 kDa to 97 kDa.ConclusionThe obtained results suggested that the mucus of this species has proteinaceous compounds responsible for antimicrobial and hemolytic activities.

Di-(2-Ethylhexyl) Phthalate Promotes Release of Tissue Factor-Bearing Microparticles From Macrophages via the TGFβ1/Smad/PAI-1 Signaling Pathway

BackgroundPlasminogen activator inhibitor type 1 promotes formation of endothelial microparticles with procoagulant activity. However, it remains unclear whether di-(2-ethylhexyl) phthalate, a peroxisome proliferator-activated receptor α agonist, influences microparticle formation. Materials and MethodsThe effect of di-(2-ethylhexyl) phthalate on release of tissue factor-bearing microparticles was investigated using human M1 macrophages. ResultsExposure of M1 macrophages to di-(2-ethylhexyl) phthalate significantly upregulated expression of plasminogen activator inhibitor type 1, whereas incubation of macrophages with small interfering RNA for peroxisome proliferator-activated receptor α attenuated it. Di-(2-ethylhexyl) phthalate significantly increased the tissue factor protein level in culture supernatants of M1 macrophages, but not M2 macrophages. After purification of proteins by centrifugal filtration, western blotting detected 2 high molecular weight bands of tissue factor-bearing microparticles in culture supernatants of M1 macrophages. The upper band showed binding to factor VIIa and tissue factor pathway inhibitor, unlike the lower band. This suggested heterogeneity of the procoagulant activity of tissue factor-bearing microparticles, presumably dependent upon encryption/decryption of tissue factor. Phosphatidylserine contributes to tissue factor decryption, and western blotting revealed that the density of phosphatidylserine was reduced in the upper tissue factor band compared with the lower band. Di-(2-ethylhexyl) phthalate also upregulated transforming growth factor-β1 protein production by M1 macrophages. Moreover, silencing of Smad2, Smad3 or Smad4 attenuated plasminogen activator inhibitor type 1 expression and tissue factor-release from macrophages after di-(2-ethylhexyl) phthalate stimulation. ConclusionsDi-(2-ethylhexyl) phthalate promotes formation of tissue factor-bearing microparticles in human M1 macrophages via the transforming growth factor-β1/Smad/ plasminogen activator inhibitor type 1 signaling pathway.

Redox of Cysteines and Protein Folding of Snap-25

The SNARE proteins SNAP-25, syntaxin, and VAMP play critical roles in neuronal exocytosis by providing the four helical regions (SNARE domains) that form a coiled-coil complex required for neurotransmitter release. This complex is continually formed and unwound as exocytotic vesicles fuse and are recycled. Using multiple techniques, we observe clear structural changes in SNAP-25B due to temperature, pH, ionic strength, methanol, and oxidation state. Here we focus on redox state and structural changes due to temperature and methanol. Using Circular Dichroism to measure the secondary structure of SNAP-25B in low ionic strength solutions, we observe an increase in alpha-helical structure when methanol is added, or the temperature is lowered, similar to the helical shift observed when SNAP-25 forms a complex with syntaxin and VAMP. Methanol (30%) or heating (34C) also breaks up large aggregates of SNAP-25B, as observed by decreased opacity in the CD signal and a loss of high molecular weight bands in non-denaturing gels. The four cysteines in SNAP-25B are thought to be palmitoylated in vivo and can be modified by addition of iodoacetic acid (IAA). Using non-denaturing gels, we observe a shift of mobility of SNAP-25B depending on the redox potential of the reaction solution. We previously showed that these cysteines are oxidized by micromolar Cu2+ by measuring the number of cysteines accessible for labeling with a Biotin-maleimide tag technique developed in our lab (Woodbury et al., 2011. Anal. Biochem. 417:165-173). These data support the hypothesis that SNAP-25B structure and cysteine accessibility are sensitive to environmental changes, which may modulate SNAP-25 palmitoylation and neurosecretion in vivo.

Cloning, expression and characterization of a novel alkaline serine protease gene from native Iranian Bacillus sp.; a producer of protease for use in livestock

The use of proteases in the last decade has been welcomed in livestock and poultry industries and has led to significant results such as improved feed conversion ratio, weight gain and increased growth performance. In the present study, isolation and identification of a novel alkaline protease from Iranian Bacillus species was performed in order to use in livestock feed. After primary isolation of bacteria from soil samples of rice fields and early detection of bacterial genus, the zymogram plate was performed for evaluation of production extracellular proteases. Of the 11 strains producing protease, one strain that produced more enzymes was selected to continue the work. Characterization of alkaline protease was done using specific enzyme assays. To confirm the genus of isolates as well as to identify the species close to, molecular analysis of 16S rRNA gene sequence was done. After that, bioinformatics analysis carried out in NCBI database for searching bacterial alkaline proteases gene sequences. The primer designed based on gene homology of close species for extraction of alkaline protease gene. The results showed that the enzyme extract had the highest activity at pH 9.0 and 50 °C. The 16S rRNA gene sequence was submitted for the strain called Bacillus sp. RAM on the NCBI site. According to the results of the phylogenetic tree, the bacterium was belonged to Bacillus genus and Bacillus sp. RAM was close to Thuringiensis C405. The isolated alkaline protease gene successfully cloned in pET28a and transferred to the expression host E.coli BL21. The expression of the protease gene was evaluated by SDS-PAGE electrophoresis. The induced recombinant cells expressed the protease and revealed molecular weight band of about 38 kDa. According to the enzyme properties, this alkaline protease can useful for application in animal industry.

Localized incorporation of outer membrane components in the pathogen Brucella abortus.

The zoonotic pathogen Brucella abortus is part of the Rhizobiales, which are alpha-proteobacteria displaying unipolar growth. Here, we show that this bacterium exhibits heterogeneity in its outer membrane composition, with clusters of rough lipopolysaccharide co-localizing with the essential outer membrane porin Omp2b, which is proposed to allow facilitated diffusion of solutes through the porin. We also show that the major outer membrane protein Omp25 and peptidoglycan are incorporated at the new pole and the division site, the expected growth sites. Interestingly, lipopolysaccharide is also inserted at the same growth sites. The absence of long-range diffusion of main components of the outer membrane could explain the apparent immobility of the Omp2b clusters, as well as unipolar and mid-cell localizations of newly incorporated outer membrane proteins and lipopolysaccharide. Unipolar growth and limited mobility of surface structures also suggest that new surface variants could arise in a few generations without the need of diluting pre-existing surface antigens.

Effectiveness of daily directly observed treatment, short-course regimen among patients registered for treatment at an urban primary health center in Bengaluru

Background: Tuberculosis (TB) is a major public health problem in India with high morbidity and mortality. As per the World Health Organization guidelines, the Revised National Tuberculosis Control Program introduced daily directly observed treatment, short-course (DOTS) regimen with a fixed-dose combination with weight bands. This study was undertaken to compare the effectiveness of daily DOTS regimen with intermittent regimen and to assess the proportion of adverse drug reactions in both groups. Materials and Methods: A descriptive study was conducted at a peripheral health institute under one of the TB Units in South Bengaluru. Participants registered for treatment during the third and fourth quarter of 2017 were selected using continuous sampling. Data were collected by case record analysis, structured interviews, and telephonic follow-up. Results: The study included 81 participants, with the mean age of 40 ± 16.1 years. Majority of the study participants 55 (67.9%) were male, and majority (38 [46.9%]) belonged to the upper-lower class. Forty-two (51.8%) of the study participants were on intermittent regimen, and 39 (48.1%) were on daily DOTS regimen. There was 100% sputum conversion at the end of treatment under both treatment regimens. A total of 36 (85.7%) participants under intermittent regimen and nine (23%) under daily regimen developed one or the other adverse drug reactions. The treatment success for participants under intermittent regimen was 38 (90.47%) and that for daily regimen was 35 (89.74%). However, there was no statistically significant difference between the two groups. Conclusion: Both daily and intermittent DOTS regimens are equally effective in TB treatment, but adverse drug reactions were more common with the intermittent regimen.

Equivalent circuit model and microwave reflection loss mechanism of double-layer spiral-ring metasurface embedded composite microwave absorber

High-performance absorbing material can play an important role in electromagnetic compatibility, electromagnetic radiation protection, and anti-detection of special equipment. Combining traditional absorbing material with metamaterial is an important direction for developing absorbing material. The composite absorbing body based on the development of metamaterial has advantages of thin thickness, light weight, strong absorption, and adjustable absorption band, but the super material absorption body composed of single-sized metal pattern elements possesses generally strong absorption only for electromagnetic waves at a certain frequency. It is difficult to meet the requirement for wide frequency absorption in practical applications. In order to broaden the absorption bandwidth of metamatial, metal spiral-ring metasurface coated short carbon fiber absorber with enhanced microwave absorbing performance is proposed. The absorber is a two-dimensional structure formed by periodically arranging a large number of individual absorber units in the horizontal and vertical direction. In the HFSS simulation software, a " master-slave boundary condition” consisting of " master boundary” and " slave boundary” is provided. Under this boundary condition, the electric field between adjacent boundaries has a phase difference, which can be used to simulate an infinite array. The research results show that the obvious enhancement of both the absorption peak and bandwidth can be observed by embedding the double-layer spiral-ring metasurfaces. The increase of initial length of spiral-rings and thickness of absorber are beneficial to further enhancing the microwave absorption. The reflection loss from 9.2 GHz to 18.0 GHz are under –10 dB (the bandwidth reaches 8.8 GHz), and the peak of &lt;i&gt;S&lt;/i&gt;&lt;sub&gt;11&lt;/sub&gt; is –14.4 dB. Besides, we find that the effective electromagnetic parameters and impedance of spiral-ring metasurface embedded microwave absorber present obvious resonant phenomenon at multi-frequencies by calculating &lt;i&gt;S&lt;/i&gt; parameters. Furthermore, an equivalent circuit model regarding double-layer spiral-ring embedded absorber is established to reveal the attenuation mechanism of microwave energy. The resonant frequencies derived from this model are well accord with the simulated results. Thereby, the multi-electromagnetic resonant frequencies make the composite microwave absorber combined with double-layer metal spiral-ring and carbon fiber have microwave reflection loss in a wide bandwidth.

Corneal keratin aggresome (CKAGG) formation and clearance by proteasome activation

PurposeTo understand the mechanism of corneal keratin expression and clearance in corneal epithelium with Limbal Stem Cell Deficiency (LSCD). The hypothesis is that LSCD-induced proteasome dysfunction is a contributing factor to keratin aggregation, causing corneal keratin aggresome (CKAGG) formation. MethodLSCD was surgically induced in rabbit corneas. LSCD corneal epithelial cells (D-CEC) were collected to investigate keratin K4 and K13 expression and CKAGG formation. Oral mucosal epithelial cells (OMECS) were isolated and cultured to study K4 and K13 expression. Cultured cells were treated with proteasome inhibitor to induce CKAGG formation. ResultsK4 and K13 were strongly expressed in D-CEC, with additional higher molecular weight bands of K4 and K13, suggesting CKAGG formation. Double staining of K4/K13 and ubiquitin showed co-localization of these keratins with ubiquitin in D-CEC. Proteasome inhibition also showed K4/K13 modification and accumulation in cultured OMECS, similar to D-CEC. Proteasome activation was then performed in cultured OMEC. There was no accumulation of keratins, and levels of unmodified keratins were found significantly reduced. ConclusionResults showed an abnormal expression of K4 and K13 after LSCD-induced proteasome dysfunction, which coalesce to form CKAGG in Corneal Epithelial Cells (CEC). We propose that CKAGG formation may be one of the causative factors of morphological alterations in the injured corneal epithelium, and that CKAGG could potentially be cleared by enhancing proteasome activity.

Mutations in the NOTCH1 PEST Domain Enhance the Expression of CCL17 By Displacing the Transcription Factor HDAC1 in Chronic Lymphocytic Leukemia

The NOTCH1 is a ligand dependent transcription factor that plays an important role in lymphocyte differentiation and apoptosis . NOTCH1 dysfunction is closely related to the proliferation, differentiation, and apoptosis of tumor cells in chronic lymphocytic leukemia (CLL). With the popularization of next-generation sequencing technology the relationship between NOTCH1 mutations and disease progression in CLL, has attracted increasing attention. Here, we investigated whether the loss of a 2 bp frame shift deletion mutation influences the NOTCH1 pathway and whether this mutation alters the NOTCH1 nuclear mechanism in CLL.The ICN plasmid was engineered by cloning the ICN coding sequence into a pmax-Clover vector. The c.7541-7542delCT mutation (CTdel) was generated by site-specific mutagenesis. The BaF3 cells were transfected with Amaxa Nucleofector technology then sorted. The NOTCH1 protein expression was evaluated by Western blotting using an anti-NOTCH1 antibody, which showed compatible with the ICN. The CTdel mutation resulted in a lower molecular weight band, consistent with the presence of a premature STOP codon. Results from qRT-PCR showed elevated mRNA expression of NOTCH1 in the groups transfected with ICN and CTdel genes. An immunofluorescence assay showed that NOTCH1 was distributed in both the nuclei and cytoplasm in the control cells, while it was located in the nucleus of the cells of the ICN and CTdel groups. Compared with the control group, the activity of the reporter genes in both the ICN and CTdel groups increased, with the highest increase in the CTdel group, as reported by the two-fluorescent enzyme reporting system assay. These results determine the presence or absence of a NOTCH1 mutation, the ICN protein is located in the nucleus, and show that the NOTCH1 pathway is enhanced and the function is more stable in the presence of a NOTCH1 mutation. From the RNA-seq results we found that RT-PCR showed transcription levels of CCL17 in the ICN and CTdel groups were higher than those in the control group, and that CCL17 in the CTdel group was significantly higher than in the ICN group.We collected the culture supernatant of CLL cells for an ELISA assay and found that CCL17 was significantly elevated in the CTdel group, but CCL17 was not detected in the control group and the ICN group. In order to verify the CCL17 function of in CLL with the NOTCH1 mutation, we performed a transwell experiment to detect the ability of mediating activated CD4+ T cell migration by CCL17. The results showed that the number of CD4+ T-cells in the CTdel group that migrated in response to CCL17 was more than in the ICN group.In order to verify that the NOTCH1 mutation changed the ICN binding function, we performed a CO-IP experiment. The results showed that ICN had an interaction with MTA2/HDAC1, but this interaction was weakened with CTdel. Mass spectrometry (MS) analysis suggested that ICN was combined with MTA2 while CTdel peptides were not detected in MTA2 samples. In order to verify the negative regulatory effect of MTA2 and HDAC1 on the NOTCH1 mutation induced by C-terminal truncation in CCL17 transcription, we conducted a CHIP experiment on the nuclear pyrolysis of ICN/CTdel.The results showed that the combination of MTA2/HDAC1 and the promoter of CCL17 and ICN/CTdel was weaker than that of the control group. Because of the multiple-point binding characteristics of transcription factors on gene expression regulation, it can be concluded that CTdel DNA binding is weaker than the binding of ICN. As a result, in the presence of the NOTCH1 protein C-terminal truncation, which has lost MTA2/HDAC1 binding, its inhibition is reduced and the CCL17 expression becomes significantly elevated.In conclusion, it is suggested that the NOTCH1 mutation found in CLL stimulates the NOTCH1 pathway, and is related to the high expression of CCL17. The chemokine CCL17 can cause the migration of CD4+ T-cells and change the microenvironment to favor tumor cell survival. ICN in the nucleus combines with CSL to form activating complexes or recruits transcription factor MTA2/HDAC1 to form inhibiting complexes, and constitutes the balance between the promotion and the inhibition for the downstream gene expression. The NOTCH1 mutation with CTdel could result in loss of this balance, and activate the expression of downstream genes, such as CCL17.Key words: chronic lymphocytic leukemia; NOTCH1; mutation; HDAC1; CCL17; chemokine DisclosuresNo relevant conflicts of interest to declare.

Population Pharmacokinetics of Adjunctive Lacosamide in Pediatric Patients With Epilepsy.

A pediatric population pharmacokinetic model including covariate effects was developed using data from 2 clinical trials in pediatric patients with epilepsy (SP0847 and SP1047). Lacosamide plasma concentration-time data (n = 402) were available from 79 children with body weights ranging from 6 to 76 kg, and a balanced age distribution (6 months to <2 years: n = 14; 2 to <6 years: n = 22; 6 to <12 years: n = 25; 12 to <18 years: n = 18). A single-compartment population pharmacokinetic model with first-order absorption and elimination described the data adequately. Plasma clearance was modeled using allometric scaling on body weight with a freely estimated allometric exponent, while volume of distribution used a fixed theoretical allometric exponent. Covariate search identified a significant effect of enzyme-inducing antiepileptic drugs resulting in a 35% decrease in lacosamide average plasma concentration. No additional effects on clearance could be attributed to race, sex, age, or renal function. Different dosing adaptation schemes by body weight bands were simulated to approximate, in pediatric patients aged 4 to 17 years, the same average plasma concentration as in adult patients receiving the maximum recommended lacosamide daily dose.

The Dry-aging and Heating Effects on Protein Characteristics of Beef Longissiumus Dorsi.

The aim of this study was to investigate the effects of dry-aging (DA) and the cooking process on the myofibril protein functionalities and in vitro digestibility of proteins in beef loin. Six sirloins from beef were dry-aged for 28 d, and the control group (n=6) was analyzed 2 d postmortem for this study. Dimensional changes (reduction of thickness and surface shrinkage) after cooking were significantly greater in the control group than the DA group, whereas the shear force of the DA group was significantly lower than that of the control. Effect of cooking on aggregation, hydrophobicity, and in vitro digestibility were significantly higher in the DA group than in the control. After cooking, the protein in DA sirloins was more oxidized than in the control samples. According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis result, the low molecular weight bands (below 17 kDa) increased in the DA group, finding that the protein characteristics of dry-aged beef was affected by cooking.

Identification of a perchloric acid-soluble protein (PSP)-like ribonuclease from Trichomonas vaginalis

A perchloric acid-soluble protein (PSP), named here tv-psp1, was identified in Trichomonas vaginalis. It is expressed under normal culture conditions according to expressed sequence tag (EST) analysis. On the other hand, Tv-PSP1 protein was identified by mass spectrometry with a 40% of identity to human PSP (p14.1). Polyclonal antibodies against recombinant Tv-PSP1 (rTv-PSP1) recognized a single band at 13.5kDa in total protein parasite extract by SDS-PAGE and a high molecular weight band analyzed by native PAGE. Structural analysis of Tv-PSP1, using dynamic light scattering, size exclusion chromatography, and circular dichroism spectroscopy, showed a trimeric structure stable at 7M urea with 38% α-helix and 14% β-sheet in solution and a molecular weight of 40.5kD. Tv-PSP1 models were used to perform dynamic simulations over 100ns suggesting a stable homotrimeric structure. Tv-PSP1 was located in the nucleus, cytoplasm, and hydrogenosomes of T. vaginalis, and the in silico analysis by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) showed interactions with RNA binding proteins. The preliminary results of RNA degradation analysis with the recombinant Tv-PSP1 showed RNA partial deterioration suggesting a possible putative ribonuclease function.

Expression of a 100kDa recombinant protein from Histoplasma capsulatum in the methylotrophic yeast Pichia pastoris

Background: Histoplasmosis is a systemic and endemic mycosis widely distributed in the Americas caused by the dimorphic fungus Histoplasma capsulatum. The infection is usually asymptomatic in immunocompetent individuals. However, immunocompromised patients may contract the disseminated form of the disease, which has a bad prognosis and requires rapid diagnosis and treatment. The definitive diagnosis involves the isolation of H. capsulatum by culture from clinical specimens, which may take up to 4 weeks. In addition, molecular methods are expensive and have low sensitivity and immunoassays present many false-positive results. Thus, the aim of this work is to express Hc100, a specific protein of 100kDa from H. capsulatum, to develop a novel direct immunoassay and to perform characterization studies of this protein as a first approach for the potential development of novel therapeutic strategies. Methods & Materials: The gene that codes for Hc100 was constructed with a secretory signal and a polyhistidine-tag and was expressed in Pichia pastoris X-33 strain. Cell culture supernatants and lysates from different induction times were analyzed by SDS-PAGE, Westernblot and mass spectrometry. Results: A band of the expected size was observed in the supernatants at 24, 48 and 72 h of methanol induction in Coomasie blue stained gels and its identity was confirmed by Westernblot using anti-histidine antibodies and by mass spectrometry. The highest expression level was observed at 24 h of induction. Also, a lower molecular weight band was observed at 48 and 72 h of induction, probably due to degradation processes. Conclusion: P. pastoris proved to be a valid biotechnological tool for the expression of this specific protein, thus encouragingthe national production of novel fungal antigens for the potential development of new rapid diagnostic tests for this clinical relevant form of the histoplasmosis disease.