1,340 research outputs found
Functional studies on factor VIII variants containing different lengths of the B-domain AND The construction of a new helper vector for the production of helper-dependent adenovectors
Functional studies on factor VIII variants containing different lengths of the B-domain To evaluate the role of the factor VIII (FVIII) B-domain on FVIII expression, (activity and activation), we constructed several FVIII expression constructs with various lengths of the B- domain and expressed them transiently as well as stably. Practically, B-domain deleted recombinant FVIII (BDD-rFVIII) was generated from the full- length FVIII-cDNA by ligating (on the protein level) Ser743 to Arg1634; the corresponding FVIII-cDNA was then cloned into the pMT2 expression vector. Then, cDNA-fragments of different length (on the protein level: 101, 202, 292, and 394 amino acid long fragments of the B-domain starting from its amino terminal) were cloned between Ser743 and Arg1634. Constructs were then transiently expressed in COS-7, HEK 293 and CHO cells and stably expressed in CHO-DHFR(-) cells by methotrexate selection and amplification. FVIII proteins were then assessed for activity in culture medium by the clotting assay and the chromogenic assay and for antigen in cellular lysates by ELISA. FVIII proteins were purified from stable cell lines media. Our transient expression studies showed that FVIII expression was the highest in COS-7 cells followed by HEK 293 cells. The CHO cells showed the lowest FVIII expression. Transient expression in COS-7 cells (for forty-eight hours) revealed that the constructs containing added lengths of the B-domain showed a higher FVIII: C (~1.1-2.5 folds) and a higher overall expressed protein (total antigen in medium and cellular lysates; ~1.1-3 folds) compared the BDD-rFVIII. The functionality of the expressed proteins in terms of FVIII specific activity was higher in all constructs in comparison to the BDD construct. Comparing the clotting FVIII activity by that of the chromogenic assay, we found that those constructs showed a slightly higher FVIII value within the clotting assay than within the chromogenic assay. In contrast, the BDD construct showed the opposite effect and also showed a significant discrepancy between both assay methods. Activation of purified FVIII proteins by thrombin revealed that the BDD-rFVIII showed a faster generation of the A2 domain than the constructs containing added lengths of the B-domain. In conclusion, our data showed that the inclusion of a 100 amino acids or more of the B- domain sequence eliminates the assay discrepancy and increases the overall expression and functionality of rFVIII protein.The construction of a new helper vector for the production of helper-dependent adenovectors Fully deleted Helper-Dependent Adenovectors (HD-AdV) are so far the ultimate form of Adenovirus vector modifications which lack all the adenovirus genes except the replication and packaging elements. They can accommodate an insert of up to 36 kb. HD-AdVs are produced in HEK 293 cells in the presence of a helper adenovirus that supplies the viral genes in trans. Using this system very high titers of HD-AdV can be prepared now, with small but still measurable helper adenovirus contamination. Within this study we have tried to design a novel hybrid helper virus that can support the production of a HD-AdV, carrying a B-domain-deleted factor VIII-cDNA, in high efficiency without any contamination. In order to avoid contamination of HD-AdV production, the adenoviral sequences in the novel hybrid helper virus was lacking the adenoviral ITRs and the packaging signal, but was flanked by adeno-associated virus (AAV) ITRs. This adenoviral helper sequences was then cloned into the baculovirus expression system. The lack of the Adenovirus packaging signal renders the hybrid helper virus packaging incompetent and thus eliminates the risk of contaminating the HD-AdV preparations. Since the helper virus also lacks the adenoviral ITRs, it will not compete with the HD-AdV for the Adenovirus 5 replication proteins. The lack of the capsid genes within the AAV-sequences should also prevent the generation of AAV particles. To drive the replication of the Ad5-genome in the hybrid helper virus theAAV2-Rep genes were cloned in a separate baculoviral construct. A GFP baculoviral construct was prepared to be used as positive control for transfecting insect cells and infecting HEK 293 cells. We successfully constructed the hybrid helper vector pBac-AAV2-Ad5 with a deletion in the 3’ITR region. The recombinant baculovirus particles (vBac-AAV2-Ad5, vBac-AAV2-Rep and vBac-EGFP) was then produced in insect cells, plaque purified and amplified to high titers. Since the deletion within the 3’-ITR region should have no effect on helper virus production, the produced baculoviruses were used to infect HEK 293 cells, together with the HD-AdV carrying the B-domain-deleted factor VIII-cDNA. Depending on the presence of AAV-ITRs, we expected that the genome of the helper-virus will be replicated, thus improving its efficiency in providing helper function. To verify our strategy in synthesizing the helper virus, we showed that the baculovirus is able to infect HEK 293 cells by eGFP expression and that the Rep gene was expressed in both SF9 cells and HEK 293 cells. After three rounds of infecting HEK293 cells with the helper hybrid virus, we couldn’t see any signs of HD-AdV production. Unexpectedly, the analysis of extracted extra chromosomal DNA showed that the material was the original transfected material and not synthesized within the HEK 293 cells. In conclusion, as the baculovirus was able to infect HEK 293 cells and the Rep genes were also expressed in those cells, we propose that the HD-AdV was not produced because of inefficient or non occurring replication of the Ad5 genes in the Bac/Ad5 hybrid. The presence of only one ITR may be a possible cause. The reconstruction of an helper hybrid with both ITRs would clarify the function of such an adenoviral helper virus system
Elastomer-based visuotactile sensor for normality of robotic manufacturing systems
Modern aircrafts require the assembly of thousands of components with high accuracy and reliability. The normality of drilled holes is a critical geometrical tolerance that is required to be achieved in order to realize an efficient assembly process. Failure to achieve the required tolerance leads to structures prone to fatigue problems and assembly errors. Elastomer-based tactile sensors have been used to support robots in acquiring useful physical interaction information with the environments. However, current tactile sensors have not yet been developed to support robotic machining in achieving the tight tolerances of aerospace structures. In this paper, a novel elastomer-based tactile sensor was developed for cobot machining. Three commercial silicon-based elastomer materials were characterised using mechanical testing in order to select a material with the best deformability. A Finite element model was developed to simulate the deformation of the tactile sensor upon interacting with surfaces with different normalities. Additive manufacturing was employed to fabricate the tactile sensor mould, which was chemically etched to improve the surface quality. The tactile sensor was obtained by directly casting and curing the optimum elastomer material onto the additively manufactured mould. A machine learning approach was used to train the simulated and experimental data obtained from the sensor. The capability of the developed vision tactile sensor was evaluated using real-world experiments with various inclination angles, and achieved a mean perpendicularity tolerance of 0.34°. The developed sensor opens a new perspective on low-cost precision cobot machining
Enriching student knowledge on sustainable forest management practices by forestry professionals: a look into the generic structure potential of Malaysian forestry annual reports
Despite the establishment of numerous goals related to sustainable forest management (SFM), including the setting of Goal 15 in the 2015 United Nations' Sustainable Development Goals (SDGs), to date, its implementation is seen to be problematic. Its poor implementation is often associated with ineffective management of forests areas, which poses some challenges for students to understand better the nature of forestry professions and the expectations that come with the responsibility of sustainably managing forest reserves and lands. Analysing the practices and agendas conveyed in the forestry annual report (FAR) is indispensable, especially for forestry students who wish better to understand the forestry profession upon their study completion. This study sets out to identify the generic structure potential for the Year under the Review section of the forestry annual report and its functions in portraying forestry professionals throughout ten years (2009–2018). Document analysis following a Systemic Functional Linguistics perspective on generic structure potential is adopted in this study to identify the obligatory and optional elements included in the forestry annual report as well as the functions of each element observed in the Year under Review section of FAR. The results of the study show that eleven obligatory elements constitute the generic structure of the Year under Review section of the forestry annual report, which is directly related to the ideal SFM practices that contribute to SDG Goal 15 (Protect, restore and promote sustainable use of terrestrial ecosystems, sustainably manage forests, combat desertification, and halt and reverse land degradation and halt biodiversity loss). The establishment of the generic structure potential for the Year under Review section of the Malaysian forestry annual report will help undergraduate forestry students understand the forestry profession's actual practices to equip them with knowledge in real-life settings
The Student-Authored Biomedical Publications at Alfaisal University, Saudi Arabia: a 6-year descriptive analysis
An audit of type 2 diabetes care in a Malaysian public community polyclinic
Adequate and proper diabetes care in any practice is paramount and deems to be the fundamental requirement
for good diabetes control. This is an audit of type 2 diabetes care process in a public Polyclinic, with the
objectives of studying the quality of diabetic care provided in terms of clinical and managerial performance
and also to give recommendations on improving its diabetes care delivery. The audit was done on patients’
medical records selected via systematic random sampling. Patients who have been diagnosed with type 2
diabetes mellitus for more than 2 years, and those who have come for follow-up at least twice in between 1st
October 2008 and 30th September 2009 by the medical officer and/or family medicine specialist. Patients with
gestational diabetes mellitus and Type 1 diabetes mellitus were excluded. A total of 100 medical records were
audited. 51% were female and 82% were in the 40-69 age group. Measurements of blood glucose and blood
pressure were done at each follow-up at the polyclinic at 96% and 93% of the times, respectively. Within the
past one year, HbA1c was performed in 46% of the patients, while renal profile was screened in 66% of the
patients and urine protein was tested in 59% of the diabetics. Only 15% had their eyes screened by fundus
camera. The diabetes care process at this public polyclinic was unsatisfactory, as many annual blood tests
were not done and complications screening were also omitted. Thus, an urgent intervention is recommend in
order to rectify these inadequacies
Consensus evidence-based recommendations for treat-to-target management of immunoglobulin A vasculitis
IgA vasculitis (IgAV), formerly known as Henoch-Schönlein purpura, is the most common cause of systemic vasculitis in childhood. Given its potential life-threatening systemic
complications, early and accurate diagnosis as well as management of IgAV represent a major challenge for health care professionals. This study was carried out to attain an evidence-based expert consensus on a treat-to-target management approach for IgAV using Delphi technique.
The preliminary scientific committee identified a total of 16 key clinical questions according to the patient, intervention, comparison, and outcomes (PICO) approach. An evidence-based, systematic, literature review was conducted to compile evidence for the IgAV management.
The core leadership team identified researchers and clinicians with expertise in IgAV management in Egypt upon which experts were gathered from different governorates and health centers across Egypt. Delphi process was implemented (two rounds) to reach a consensus. An online questionnaire was sent to expert panel (n = 26) who participated in the two rounds. After completing round 2, a total of 20 recommendation items, categorized into two sections were
obtained. Agreement with the recommendations (rank 7–9) ranged from 91.7–100%.
Consensus was reached (i.e. ⩾75% of respondents strongly agreed or agreed) on the wording of all the 20 clinical standards identified by the scientific committee. Algorithms for the diagnosis and management have been suggested. This was an expert, consensus recommendations for the diagnosis and treatment of IgAV and IgA vasculitic nephritis, based on best available evidence and expert opinion. The guideline presented a strategy of care with a pathway to achieve a state of remission as early as possible
Social support for students with visual impairments in educational institutions : an integrative literature review
Students with visual impairments often experience emotional problems and encounter difficulties in forming and maintaining social relationships. Research indicates that the social support provided to these students by staff members and their peers in educational institutions may have a positive impact on their academic learning and socioemotional development. The purpose of this integrative literature review was to synthesise the results from 17 academic articles published during 1998 and 2018, which examined the topic of social support for students with visual impairments in educational institutions. This review reveals that for students with visual impairments cooperation, empathetic behaviour, and practical assistance are the main components of social support. These students actively seek social support from staff members and peers, but they face many challenges, such as the lack of training and awareness. Support from staff members contributes to students’ academic learning and social inclusion, whereas peers’ social support enhances their self-esteem and social acceptance. The outlined positive effects of educational interventions on students’ social skills and social interaction support the need for implementing more interventions. The limitations of the studies reviewed and recommendations for future research are discussed
Ultrasonic Relaxation of TeWB Glasses at Low Temperatures
The ultrasonic relaxation of tellurite glasses xB2O3 – 70 TeO2 – (30 – x) WO3, 0 ≤ x ≤ 30 mol% was investigated by measuring the ultrasonic attenuation (αL) in these glasses in the thermal range 140–300 K (T) at various frequencies (f). Some characteristics can be obtained from the relation of αL–T, such as the average activation energy (Ep) and the attempt frequency (f0). The variables Ep and f0 are a function of frequency and B2O3 content. Moreover, the relation αL–T accounts well for the oscillations of the oxygen atoms in a relaxation process. Such a physical process is originated from transmitting ultrasonic energy to the oscillating oxygen atoms in a dual-well potential. The relaxation process is inspected by a model named central force one. The subtracted different physical variables from such a model are a function of B2O3 content. © 2021 The Author(s).The authors express their gratitude to the Deanship of Scientific Research, Majmaah University, Saudi Arabia, for funding this research work under Grant (R-2021-125)
Characterization of 1577 primary prostate cancers reveals novel biological and clinicopathologic insights into molecular subtypes.
BACKGROUND: Prostate cancer (PCa) molecular subtypes have been defined by essentially mutually exclusive events, including ETS gene fusions (most commonly involving ERG) and SPINK1 overexpression. Clinical assessment may aid in disease stratification, complementing available prognostic tests.
OBJECTIVE: To determine the analytical validity and clinicopatholgic associations of microarray-based molecular subtyping.
DESIGN, SETTING, AND PARTICIPANTS: We analyzed Affymetrix GeneChip expression profiles for 1577 patients from eight radical prostatectomy cohorts, including 1351 cases assessed using the Decipher prognostic assay (GenomeDx Biosciences, San Diego, CA, USA) performed in a laboratory with Clinical Laboratory Improvements Amendment certification. A microarray-based (m-) random forest ERG classification model was trained and validated. Outlier expression analysis was used to predict other mutually exclusive non-ERG ETS gene rearrangements (ETS(+)) or SPINK1 overexpression (SPINK1(+)).
OUTCOME MEASUREMENTS: Associations with clinical features and outcomes by multivariate logistic regression analysis and receiver operating curves.
RESULTS AND LIMITATIONS: The m-ERG classifier showed 95% accuracy in an independent validation subset (155 samples). Across cohorts, 45% of PCas were classified as m-ERG(+), 9% as m-ETS(+), 8% as m-SPINK1(+), and 38% as triple negative (m-ERG(-)/m-ETS(-)/m-SPINK1(-)). Gene expression profiling supports three underlying molecularly defined groups: m-ERG(+), m-ETS(+), and m-SPINK1(+)/triple negative. On multivariate analysis, m-ERG(+) tumors were associated with lower preoperative serum prostate-specific antigen and Gleason scores, but greater extraprostatic extension (p
CONCLUSIONS: A clinically available prognostic test (Decipher) can also assess PCa molecular subtypes, obviating the need for additional testing. Clinicopathologic differences were found among subtypes based on global expression patterns.
PATIENT SUMMARY: Molecular subtyping of prostate cancer can be achieved using extra data generated from a clinical-grade, genome-wide expression-profiling prognostic assay (Decipher). Transcriptomic and clinical analysis support three distinct molecular subtypes: (1) m-ERG(+), (2) m-ETS(+), and (3) m-SPINK1(+)/triple negative (m-ERG(-)/m-ETS(-)/m-SPINK1(-)). Incorporation of subtyping into a clinically available assay may facilitate additional applications beyond routine prognosis
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