Weight Bands Research Articles

Glycosylated Chromogranin A in Heart Failure: Implications for Processing and Cardiomyocyte Calcium Homeostasis.

Chromogranin A (CgA) levels have previously been found to predict mortality in heart failure (HF), but currently no information is available regarding CgA processing in HF and whether the CgA fragment catestatin (CST) may directly influence cardiomyocyte function. CgA processing was characterized in postinfarction HF mice and in patients with acute HF, and the functional role of CST was explored in experimental models. Myocardial biopsies from HF, but not sham-operated mice, demonstrated high molecular weight CgA bands. Deglycosylation treatment attenuated high molecular weight bands, induced a mobility shift, and increased shorter CgA fragments. Adjusting for established risk indices and biomarkers, circulating CgA levels were found to be associated with mortality in patients with acute HF, but not in patients with acute exacerbation of chronic obstructive pulmonary disease. Low CgA-to-CST conversion was also associated with increased mortality in acute HF, thus, supporting functional relevance of impaired CgA processing in cardiovascular disease. CST was identified as a direct inhibitor of CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, and CST reduced CaMKIIδ-dependent phosphorylation of phospholamban and the ryanodine receptor 2. In line with CaMKIIδ inhibition, CST reduced Ca2+ spark and wave frequency, reduced Ca2+ spark dimensions, increased sarcoplasmic reticulum Ca2+ content, and augmented the magnitude and kinetics of cardiomyocyte Ca2+ transients and contractions. CgA-to-CST conversion in HF is impaired because of hyperglycosylation, which is associated with clinical outcomes in acute HF. The mechanism for increased mortality may be dysregulated cardiomyocyte Ca2+ handling because of reduced CaMKIIδ inhibition.

Compact and Low-Profile Omnidirectional Circularly Polarized Antenna With Four Coupling Arcs for UAV Applications

A compact and low-profile omnidirectional circularly polarized (CP) antenna with four coupling arcs is presented. In this structure, four outer arcs are utilized to generate horizontal polarization, and an inner disk-loaded feeding pin can produce vertical polarization. Thus, omnidirectional CP radiation is achieved by combining the radiation from the arcs and the feeding pin. Four shorting pins are employed at the end of the outer arcs, which play a great role on the miniaturization of the antenna. Furthermore, the antenna's dimensions can be reduced more by loading four inner coupling arcs inside the outer arcs. An L-band prototype of the antenna is calculated, fabricated, and measured. Its final dimensions are $0.16{\lambda _0} \times 0.16{\lambda _0} \times 0.03{\lambda _0}$ ( ${\lambda _0}$ is the free-space wavelength at operating frequency). The results show the impedance bandwidth (VSWR ≤2) of 13 MHz (1429–1442 MHz). In the operating frequency band, the axial ratios are less than 2.4 dB, and the peak gain reaches 1.01 dBic. This type of antenna can meet the requirements of the weight, dimensions, and frequency bands for special unmanned aerial vehicle communications in China.

P1.02-036 An EGFR Tyrosine Kinase Inhibitor Sensitive Patient-Derived Lung Cancer Xenograft Model without Classical Sensitizing Mutations

Mutations in the tyrosine kinase (TK) domain of EGFR are oncogenic driver in 10-20% of lung adenocarcinoma (AdC) patients in Western countries. Approximately 90% of EGFR-TK inhibitor (TKI) sensitizing mutations occur as small in-frame deletions in exon 19 or L858R point mutations in exon 21. Recently, novel driver mutations in EGFR with oncogenic and TKI sensitizing activity have been reported. We present here an AdC patient-derived xenograft (PDX) model (PDX12) that is highly sensitive to EGFR-TKI, yet failed to demonstrate classical TKI sensitizing mechanisms. Comprehensive genomics profiling was used to characterize the genotype of PDX12, which was established from a resected stage IIIA AdC patient grafted in NGS mouse. The primary human lung cancer cell line (PHLC12) was extracted from its PDX model (PDX12). Aberrant EGFR cell lines used were H3255 (L858R), H2935 (exon 19 deletion), H1975 (L858R and T790M), and H1944 (wild type). Cell viability was assessed after erlotinib treatment at 1nM - 2μM for 72 hours using MTS assay. Levels of EGFR activation in both pre- and post-treatment by Western blot analysis. PDX12 model had no known oncogenic mutations (EGFR wild type) on exons 18-21 by next-generation sequencing, RT-qPCR, and SISH, but was highly sensitive to EGFR-TKI. The IC50 to erlotinib treatment at 72 hr was 67.13 ± 7.63 nM for PHLC12, compared to 9.70 ± 2.64 nM for H3255, 64.88 ± 8.49 nM for HCC2935, > 2 μM for H1975, and > 2 μM for H1944 EGFR mutant or wild type cells, respectively. Western blot analysis demonstrated a relatively higher molecular weight band for EGFR protein with high expression level in PHLC12 when compared to other lung cancer cell lines. Using RT-qPCR, relative expression level of each EGFR domain (extracellular, tyrosine kinase, and c-terminal domain) in PHLC12 showed no difference compared to EGFR wild type. Phosphorylation status of EGFR in PHLC12 was similar in activity as compared to erlotinib sensitive cell lines. PHLC12 represents an enigmatic EGFR TKI sensitive lung PDX model without classical TKI sensitizing aberrations. Additional potential mechanisms of EGFR dependency including exon duplication, or post-translational modification of EGFR protein are being investigated.

열응답 실험 및 열저항 해석을 통한 장심도 수직밀폐형 지중열교환기의 성능 분석

300 m 이상의 장심도 지중열교환기는 도심지나 넓은 부지를 확보가기 어려운 지역에 지열냉난방 시스템을 경제적으로 설치하는데 유리하다. 그러나 실제 시공에서는 여러 가지 문제들로 인하여 보편적으로 시도되지 않았고, 일반적으로 100 ~ 200m 심도로 설치되어 왔다. 본 연구에서는 일반적인 시추공 직경 150 mm에 U 파이프는 50A 규격으로 외경 50 mm의 300 m 심도로 지중열교환기를 설치하였다. 고밀도 PE관은 단위 길이당 비중이 <TEX>$0.94{\sim}0.96g/cm^3$</TEX>으로 지열공 내부에 채워진 지하수 영향으로 부력이 존재하여, 이를 개선하기 위해 4.6 kg 무게의 금속으로 제작된 하중밴드 10개조를 설치하여 부력의 영향을 감소시켰다. 지중열교환기의 길이 산정 및 성능평가를 위한 기초조사로서 지반조사 및 열응답실험이 실시되었다. 지반내 온도구배는 100 m 심도까지는 주변 지하수 이용에 의한 영향 등으로 <TEX>$15^{\circ}C$</TEX> 정도의 분포를 보이며 그 하부는 <TEX>$1.9^{\circ}C/100m$</TEX>의 지온증온율을 나타내고 있다. 열응답실험은 기존에 설정된 표준 방식으로 48 시간 진행되었으며 평균 주입전력은 17.5 kW이며 평균 순환수 유량은 28.5 l/min, 그리고 평균 입출구 온도차는 <TEX>$8.9^{\circ}C$</TEX>로 나타났다. 측정된 지중열전도도는 3.0 W/mk이며, 공내열저항은 0.104 mk/W로 나타났다. Stepwise 평가에서 지중열전도도 변화는 초기 13시간을 제외한 이후에는 표준편차가 0.16으로 매우 안정된 값으로 수렴한 것으로 나타났다. 그리고 공내열저항의 민감도를 분석한 결과 파이프의 구경과 그라우팅 물질의 열전도도가 증가함에 따라 그 값이 미미하게 감소하는 경향을 나타내었다. Due to the limited areal space for installation, borehole heat exchangers (BHEs) at depths deeper than 300 m are considered for geothermal heating and cooling in the urban area. The deep vertical closed-loop BHEs are unconventional due to the depth and the range of the typical installation depth is between 100 and 200 m in Korea. The BHE in the study consists of 50A (outer diameter 50 mm, SDR 11) PE U-tube pipe in a 150 mm diameter borehole with the depth of 300 m. In order to compensate the buoyancy caused by the low density of PE pipe (<TEX>$0.94{\sim}0.96g/cm^3$</TEX>) in the borehole filled with ground water, 10 weight band sets (4.6 kg/set) were attached to the bottom of U-tube. A thermal response test (TRT) and fundamental basic surveys on the thermophysical characteristics of the ground were conducted. Ground temperature measures around <TEX>$15^{\circ}C$</TEX> from the surface to 100 m, and the geothermal gradient represents <TEX>$1.9^{\circ}C/100m$</TEX> below 100 m. The TRT was conducted for 48 hours with 17.5 kW heat injection, 28.65 l/min at a circulation fluid flow rate indicates an average temperature difference <TEX>$8.9^{\circ}C$</TEX> between inlet and outlet circulation fluid. The estimated thermophysical parameters are 3.0 W/mk of ground thermal conductivity and 0.104 mk/W of borehole thermal resistance. In the stepwise evaluation of TRT, the ground thermal conductivity was calculated at the standard deviation of 0.16 after the initial 13 hours. The sensitivity analysis on the borehole thermal resistance was also conducted with respect to the PE pipe diameter and the thermal conductivity of backfill material. The borehole thermal resistivity slightly decreased with the increase of the two parameters.

Proteolytic Inactivation of ADAMTS13 By Activated Factor XI

Background: ADAMTS13, a plasma metalloprotease, is secreted into blood as an active enzyme that cleaves and inactivates von Willebrand factor (VWF), which binds collagen, facilitating platelet adhesion under vascular flow. Plasma ADAMTS13 has a molecular weight of 200 kDa, consisting of a metalloprotease (MET) domain, a disintegrin-like domain, a first thrombospondin type-1 repeat (TSP1) domain, a Cys-rich domain, and a spacer domain. Moreover, the C-terminal domain of ADAMTS13 contains an additional seven TSP1 repeats and two CUB domains. ADAMTS13 has been shown to adopt a natural folded conformation, allowing its CUB domains to interact with its spacer domain. This more closed conformation prohibits the functional exosite on the spacer domain from interacting with its proteolytic site on the A2 domain of VWF. In plasma, globular ADAMTS13 will associate with VWF via necessary binding of the CUB domains to the VWF D4CK fragment. Under shear stress or flow conditions, bound ADAMTS13 will unfold leading to exposure of the spacer domain exosite and ultimately increased ADAMTS13 proteolysis VWF. Without the CUB domains, ADAMTS13 does not proteolyze VWF under flow conditions. To date, it is still uncertain how ADAMTS13 activity is regulated, and what impact this has on the inactivation of VWF. The serine proteases thrombin, activated FX (FXa), and plasmin have been shown to cleave and inactivate ADAMTS13. Based on the fact that congenital factor XI deficiencies are associated with bleeding disorders and that elevated levels of FXI is an independent risk factor for deep vein thrombosis and ischemic stroke, we hypothesize that the serine protease activated FXI (FXIa) inactivates ADAMTS13 leading to platelet aggregation and thrombus formation.Aim: To determine whether FXIa is able to cleave and inactivate ADAMTS13.Methods and results: Recombinant ADAMTS13 (250 nM) was incubated with FXIa (50 nM) for selected times (0-3 hours) at 37oC before being separated by SDS-PAGE and analyzed by Coomassie blue staining, resulting in the disappearance of the ADAMTS13 band (~200 kDa) and the appearance of lower molecular weight bands under reducing conditions. The presence of aprotinin, which inhibits FXIa activity, blocked the degradation of ADAMTS13 by FXIa.Samples were analyzed by western blot to determine the cleavage site using an anti-ADAMTS13 antibody, which specifically binds the two CUB domains, and an anti-ADAMTS13 antibody which specifically binds the MET domain. ADAMTS13 has been shown to be cleaved by serine proteases, such as plasmin, thrombin and FXa. We incubated ADAMTS13 (200nM) with equivalent concentrations of plasmin, thrombin, FXa, FXIa, FXIIa or Kallikrein at 37oC over a time interval of 0-3 hours. The addition of Ca++(5 mM) was necessary for proteolytic activity of thrombin and FXa. We observed that the ability of FXIa to cleave ADAMTS13 was found to be similar to the ability of thrombin to cleave ADAMTS13. Neither FXa, kallikrein, nor FXIIa appeared to cleave ADAMTS13.The antibody against the MET domain detected a single broad band at approximately 150 kDa. When the samples were analyzed with the antibody specific for the two CUB domains, a single broad band at approximately 50 kDa was detected, suggesting that the proteolysis of ADAMTS13 by FXIa preferentially occurs near the start of the first CUB domain. Interestingly, it has been previously reported that the CUB domains are necessary for VWF strand cleavage under flow conditions.It has been shown that the cleavage of a fluorescence-quenching substrate, FRETS-VWF73, by ADAMTS13 was enhanced after CUB1-2 domain removal. We observed that after the incubation of ADAMTS13 (30nM) with FXIa (30nM) at 37oC for 3 hours, the activity of ADAMTS13 was increased. Analysis of the samples by western blot using an anti-ADAMTS13 MET antibody confirmed the generation of the 150 kDa fragment.Conclusion: Our study suggests a novel molecular link between the regulation of VWF activity and FXI through inactivation of ADAMTS13. The results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also through limiting ADAMTS13-mediated VWF inactivation. Our future studies are focused on determining the physiological role of the proteolytic removal of the CUB domains of ADAMTS13 by FXIa under flow conditions by measuring platelet aggregation. DisclosuresNo relevant conflicts of interest to declare.

Definition of the Minimal Truncated Elements Necessary for CSF3R Leukemogenic Potential

An increasing number of CSF3R cytoplasmic domain truncation (nonsense and frameshift) and missense mutations are being identified with the wide application of next generation sequencing (NGS) to leukemia patient samples. Previous studies have demonstrated oncogenic potential of some CSF3R truncation mutations seen in severe congenital neutropenia, chronic neutrophilia leukemia (CNL), and Philadelphia-negative atypical chronic neutrophilic leukemia (aCML) patients. However, full understanding of the spectrum of CSF3R cytoplasmic mutations harboring leukemogenic potential as well as the associated mechanisms of enhanced signaling remains incomplete. In the current study, we leveraged NGS data to first validate that CSF3R cytoplasmic mutations are prevalent in CNL/aCML/unclassified myeloproliferative neoplasm (8.5%, n=212) patients and to a lesser extent in AML patients (1.3%, n=378). We did not observe CSF3R cytoplasmic mutations in other hematologic malignances. Notably, 72.2% of CSF3R cytoplasmic mutations co-occur with the membrane proximal mutation, CSF3R T618I. To evaluate the leukemogenic potential of these cytoplasmic mutations, we first investigated the minimal mutated elements necessary for CSF3R leukemogenic potential. We identified that truncation mutations of the CSF3R cytoplasmic tail between T738 and Q823 all harbor leukemogenic potential. In contrast, CSF3R truncations before E700X did not respond to G-CSF and did not transform Ba/F3 cells even when combined with T618I. Truncation mutations between E700X and T738X demonstrated reduced G-CSF sensitivity, but could transform Ba/F3 cells when combined with CSF3R T618I. Surprisingly, all the cytoplasmic missense mutations (Q754A, R769H, L777F, T781I, S795R and Q823H), seen in the patient samples did not harbor leukemogenic potential. We further identified that missense mutations disrupting the di-leucine residues L776/777, S772, or ubiquitin binding lysine residues (K655, K785 and K704) demonstrated transforming capacity.Mechanistically, truncation mutations prior to Q793 are associated with delayed receptor internalization due to the disruption of the internalization motifs (amino acids 772-778 and 779-792), whereas truncation mutations between Q793 and Q823 decrease receptor degradation, which may be associated with loss of the de-phosphorylation domain (amino acids N818-F836). All truncation mutations before Q823 demonstrate altered patterns of molecular weight bands and are sensitive to the Golgi inhibitor brefeldin A, indicating that CSF3R glycosylation plays a role in the leukemogenic potential of these truncations. We also showed that K704 is a potential ubiquitin binding site, and substitution of K704A demonstrated reduced ubiquitination. Downstream, leukemogenic cytoplasmic mutations all induce sustained STAT5 activation and are sensitive to the JAK inhibitor ruxolitinib. In summary, we have defined the region of the CSF3R cytoplasmic domain in which truncating mutations exhibit the capacity for leukemogenesis. This information will be useful for evaluating the potential clinical relevance of CSF3R mutations appearing in genomic reports from patients with myeloid leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification and characterization of peptidases secreted by quahog parasite unknown (QPX), the protistan parasite of hard clams.

Quahog parasite unknown (QPX) is a protistan parasite capable of causing deadly infections in the hard clam Mercenaria mercenaria, one of the most valuable shellfish species in the USA. QPX is an extracellular parasite found mostly in the connective tissue of clam mantle and, in more severe cases of infection, other clam organs. Histopathologic examinations revealed that QPX cells within clam tissues are typically surrounded by hollow areas that have been hypothesized to be, at least in part, a result of extracellular digestion of clam proteins by the parasite. We investigated peptidase activity in QPX extracellular secretions using sodium dodecyl sulfate-polyacrylamide gels containing gelatin as a co-polymerized substrate. Multiple peptidase activity bands of molecular weights ranging from 20 to 100 kDa were detected in QPX secretions derived from a variety of culture media. One major band of approximately 35 kDa was composed of subtilisin-like peptidases that were released by QPX cells in all studied media, suggesting that these are the most common peptidases used by QPX for nutrient acquisition. PCR quantification of mRNA encoding QPX subtilisins revealed that their expression changes with the protein substrate used in the culture media. A fast protein liquid chromatography (FPLC) was used to fractionate QPX extracellular secretions. An FPLC-fraction containing a subtilisin-type serine peptidase was able to digest clam plasma proteins, suggesting that this peptidase might be involved in the disease process, and making it a good candidate for further investigation as a possible virulence factor of the parasite.

Molecular markers of putative spermatogonial stem cells in the domestic cat

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

Optimization of the strength of the efavirenz/lamivudine/abacavir fixed-dose combination for paediatric patients.

Child-friendly, low-cost, solid, oral fixed-dose combinations (FDCs) of efavirenz with lamivudine and abacavir are urgently needed to improve clinical management and drug adherence for children. Data were pooled from several clinical trials and therapeutic drug monitoring datasets from different countries. The number of children/observations was 505/3667 for efavirenz. Population pharmacokinetic analyses were performed using a non-linear mixed-effects approach. For abacavir and lamivudine, data from 187 and 920 subjects were available (population pharmacokinetic models previously published). Efavirenz/lamivudine/abacavir FDC strength options assessed were (I) 150/75/150, (II) 120/60/120 and (III) 200/100/200 mg. Monte Carlo simulations of the different FDC strengths were performed to determine the optimal dose within each of the WHO weight bands based on drug efficacy/safety targets. The probability of being within the target efavirenz concentration range 12 h post-dose (1-4 mg/L) varied between 56% and 60%, regardless of FDC option. Option I provided a best possible balance between efavirenz treatment failure and toxicity risks. For abacavir and lamivudine, simulations showed that for option I >75% of subjects were above the efficacy target. According to simulations, a paediatric efavirenz/lamivudine/abacavir fixed-dose formulation of 150 mg efavirenz, 75 mg lamivudine and 150 mg abacavir provided the most effective and safe concentrations across WHO weight bands, with the flexibility of dosage required across the paediatric population.

Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris.

Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475mg/L with a high volumetric productivity of 4.39mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni-NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40% α-helix, 12% β-sheets with an emission maxima at 343nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.

Physicochemical and functional properties of acid precipitated soy proteins-maltodextrin conjugates.

Physicochemical and functional properties of acid precipitated soy proteins-maltodextrin conjugates.

CREATING AND AUDITING A NEW ELECTRONIC CONTINUOUS INFUSION PRESCRIPTION CHART FOR A PAEDIATRIC CRITICAL CARE UNIT

IntroductionPrescription errors, including continuous infusion prescriptions are one major source of concern in the paediatric population. Evidence suggests that use of an electronic or web-based calculator could minimise these errors....

Standard concentration infusions in paediatric intensive care: the clinical approach.

To identify the most appropriate clinical concentrations required by our paediatric intensive care unit (PICU) population with regard to accuracy of delivery and overall fluid allowance. Firstly a matrix was used to balance the concentration, dose and infusion volume (weight range 1.5-50 kg). Results were further refined considering: patient fluid allowance based on fluid volume targets, infusion pump accuracy and challenging each infusion against clinical scenarios requiring administration of multiple drug infusions found in PICU. Consideration was given to the standard concentrations routinely used in adults, in order to assess whether alignment with paediatrics was possible for some of the concentrations proposed. Finally a risk assessment of the infusions was conducted using the NPSA 20 tool. Twenty-five drugs identified as the most commonly used intravenous infusions in the unit. For the majority of the medicines, three weight bands of standard concentrations were necessary to cover the children's weight ranges and kept within predefined fluid requirements and accuracy of delivery. This work shows a patient focused systematic approach for defining and evaluating standardised concentrations in intensive care children.

HIV-1 Coinfection Does Not Reduce Exposure to Rifampin, Isoniazid, and Pyrazinamide in South African Tuberculosis Outpatients.

There are contrasting data in the literature about antituberculosis plasma drug concentrations in HIV-1-coinfected patients. We report the pharmacokinetics of rifampin, isoniazid, and pyrazinamide in a cohort of patients being treated for active tuberculosis, the majority of whom were coinfected with HIV-1 and had commenced antiretroviral therapy within 2 months of starting antituberculosis treatment. We also examined the association between antituberculosis drug concentrations and reported drug side effects at the 2-month clinical review. One hundred patients with pulmonary tuberculosis (65% coinfected with HIV-1) were intensively sampled to determine rifampin, isoniazid, and pyrazinamide plasma concentrations after 7 to 8 weeks of a daily quadruple-therapy regimen dosed according to World Health Organization (WHO) weight bands. Pharmacokinetic parameters were determined for each patient by using nonlinear mixed-effects models. HIV-1-coinfected patients had lower clearance rates for rifampin (21% decrease) and isoniazid (23% decrease) than HIV-1-uninfected patients, with resulting higher areas under the concentration-time curve from 0 to 24 h (AUC0–24) and maximum concentrations of drug in serum (Cmax). Antiretroviral therapy (ART) that included double-standard-dose lopinavir/ritonavir further lowered rifampin clearance, by 46%, and increased the AUC0–24. The current uniform dosing (per kilogram of body weight) across WHO weight bands was associated with a trend of decreased pharmacokinetic exposures for the lowest weight band. Use of fat-free mass as opposed to total body weight for allometric scaling of clearance significantly improved the model. Ambulant HIV-1-coinfected patients, the majority of whom were coprescribed ART, did not have reduced antituberculosis drug concentrations compared to HIV-1-uninfected patients.

Regulation of Neurospora Catalase-3 by global heterochromatin formation and its proximal heterochromatin region

Catalase-3 (CAT-3) constitutes the main catalase activity in growing hyphae of Neurospora crassa, and its activity increases during exponential growth or is induced under different stress conditions. Although extensive progress has been made to identify catalase regulators, the regulation mechanism of CAT-3 at the chromatin level still remains unclear. Here, we aim at investigating the molecular regulation mechanisms of cat-3 at the chromatin level. We found that CAT-3 protein levels increased in mutants defective in proper global heterochromatin formation. Bioinformatics analysis identified a 5-kb AT-rich sequence adjacent to the cat-3 promoter as a heterochromatin region because of its enrichment of H3K9me3 and HP1. Expression of CAT-3 was induced by H2O2 treatment in wild-type and such change occurred along with the accumulation of histone H3 acetylation at 5-kb heterochromatin boundaries and cat-3 locus, but without alteration of its H3K9me3 repressive modification. Moreover, disruption of 5-kb heterochromatin region results in elevated cat-3 expression, and higher levels of cat-3 expression were promoted by the combination with global heterochromatin defective mutants. Interestingly, the molecular weight and activity bands of CAT-3 protein are different in heterochromatin defective mutants compared with those in wild-type, suggesting that its N-terminal processing and modification may be altered. Our study indicates that the local chromatin structure creates a heterochromatin repressive environment to repress nearby gene expression.

VIA HOLE - DUAL RECTANGULAR SLOTTED MICROSTRIP PATCH ANTENNA (MPA) DESIGN FOR RADIO ASTRONOMY, SATELLITE, CIVIL AND MILITARY APPLICATONS

In this paper, via hole drilled microstrip patch antenna employing two rectangular slots on patch, FR4 substrate having thickness of 1.6mm and dielectric constant of ɛr = 4.4 has been proposed. The proposed antenna has been designed and analysed using Computer Simulation Technology (CST) Microwave Studio 2014. The proposed antenna has compact size, light weight and wide band operation. The via hole slot and rectangular slots have been cut in the proposed antenna in order to increase the return loss and impedance bandwidth of the antenna. The proposed antenna ground and patch has been designed using copper of thickness 0.1mm. It has been analysed that the proposed antenna has an impedance bandwidth of 725MHz with an operating frequency range of 2.6787-3.4038GHz and is resonant at 2.905 GHz. The gain, directivity and return loss of the proposed antenna are 4.52dB,4.011dBi and -72.93dB at resonant frequency of 2.905GHz, respectively. The proposed antenna can be suitably employed for Aeronautical, Weather Radar applications (2700MHZ-2900MHz), Civil and Military applications (2900MHz-3100MHz) and Satellite applications (3100MHz-3300MHz.)

Ribosomal protein S3 (rpS3) secreted from various cancer cells is N-linked glycosylated.

Ribosomal protein S3 (rpS3) is a 243 amino acid component of the 40S ribosomal small subunit. It has multiple roles in translation and extra-ribosomal functions like apoptosis and DNA repair. RpS3 is secreted only in cancer cell lines. Presently, mass spectrometry analysis revealed rpS3 to be glycosylated at the Asn165 residue. A point mutation at this residue decreased secretion of rpS3 in cancer cell lines. Secretion was also inhibited by the endoplasmic reticulum (ER)-Golgi transport inhibitor Brefeldin A and by Tunicamycin, an inhibitor of N-linked glycosylation. N-linked glycosylation of rpS3 was confirmed as necessary for rpS3 secretion into culture media via the ER-Golgi dependent pathway. RpS3 bound to Concanavalin A, a carbohydrate binding lectin protein, while treatment with peptide-N-glycosidase F shifted the secreted rpS3 to a lower molecular weight band. In addition, the N165G mutant of rpS3 displayed reduced secretion compared to the wild-type. An in vitro binding assay detected rpS3 homodimer formation via the N-terminal region (rpS3:1–85) and a middle region (rpS3:95–158). The results indicate that the Asn 165 residue of rpS3 is a critical site for N-linked glycosylation and passage through the ER-Golgi secretion pathway.

Controllable synthesis and enhanced microwave absorbing properties of Fe3O4/NiFe2O4/Ni heterostructure porous rods

We developed a coordinated self-assembly/precipitate transfer/sintering method that allows the controllable synthesis of Fe3O4/NiFe2O4/Ni heterostructure porous rods (HPRs). A series of characterizations confirms that changing [Ni2+] can effectively control the crystal size, internal strain, composition, textural characteristics, and properties of HPRs. Molar percentages of Ni and NiFe2O4 in HPRs increase with [Ni2+] in various Boltzmann function modes. Saturation magnetization Ms and coercivity Hc show U-shaped change trends because of crystal size, composition, and interface magnetic coupling. High magnetic loss is maintained after decorating NiFe2O4 and Ni on the surface of Fe3O4 PRs. Controlling the NiFe2O4 interface layers and Ni content can improve impedance matching and dielectric losses, thereby leading to lighter weight, stronger absorption, and broader absorption band of Fe3O4/NiFe2O4/Ni HPRs than Fe3O4 PRs. An optimum EM wave absorbing property was exhibited by Fe3O4/NiFe2O4/Ni HPRs formed at [Ni2+]=0.05M. The maximum reflection loss (RL) reaches −58.4dB at 13.68GHz, which corresponds to a 2.1mm matching thickness. The absorbing bandwidth (RL≤−20dB) reaches 14.4GHz with the sample thickness at 1.6–2.4 and 2.8–10.0mm. These excellent properties verify that Fe3O4/NiFe2O4/Ni HPRs are promising candidates for new and effective absorptive materials.

English

The present study was conducted to characterize the genetic diversity of 10 germplasm accessions of finger millet (Eleusine coracana (L) Gaertn) collected from diverse locations spread across the geographical zone of Northern Nigeria.&nbsp; The digest was carried out using the molecular marker Restriction Fragment Digest (RFD on agarose gel while EcoR1 and Hind III restriction enzymes were used to cut the genomic DNA at specific sites. The results of our findings using RFD generated four clear DNA bands of molecular weights ranging from 10000, 8500, 1000 and 200 KDA. This clearly suggests the existence of polymorphism among the plant accessions.&nbsp; These results demonstrate the high variability that exists amongst the genetic traits for these germplasm accessions. Information on this plant is very useful in unraveling the pedigree genetic relationships, as well as, in designing breeding and selection experiments for improvement of this crop. It will further stimulate growing interest on the genetic diversity and classification of this crop. &nbsp; Key words: Genotypic, restriction digestion, finger millet, Eco R1, Hind 111, northern Nigeria.

Engineering Periodic shRNA for Enhanced Silencing Efficacy.

RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.