CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants

Mesenchymal and stemness transdifferentiation via in-vitro infection of T24 cell line with Klebsiella pneumoniae

أثبتت الدراسات الحديثة أن وجود البكتيريا داخل أنسجة الورم له أثرعلى بيئة الورم وبالتالي يؤثرفى سلوك السرطان وتطوره واستجابة الخلايا  للعلاج. فى هذا البحث تم دراسة التحول التمايزى لخلايا سرطان المثانة الظاهرية الى خلايا ميزنكيمية و خلايا جذعية نتيجة لاحداث العدوى البكتيرية. وذلك من خلال إحداث عدوى لسلالة خلايا سرطان المثانة البولية (T24) ببكتيريا الكلبسيلا الرئوية لمدة يومين وأربعة أيام. تم قياس التعبير الجينى باستخدام جهاز البلمرة المتسلسل.وأظهرت النتائج زيادة في صفات الخلايا الميزنكيمية ؛ فزاد التعبير الجينى لجين الفيمنتين ، ونقص التعبيرالجينى للسيتوكيراتين، وعزز تحليل كيمياء الهيستولوجية المناعية هذه النتيجة. علاوة على ذلك، زاد التعبيرالجينى للجينات الدالة على الخلايا الجذعية. العدوى البكتيرية للخلايا السرطانية قد تسبب التمايز الخلوى، مما قد يؤدى الى تحسن قدرة الخلايا السرطانية على الانتشار والتجدد الذاتي. وبالتالي، قد تساهم البكتيريا في تطور سرطان المثانة وإنتشاره.Klebsiella pneumoniae has been found in the urinary tract of some bladder cancer patients. Bacterial presence within tumor tissue may affect the tumor-microenvironment and consequently influence cancer behavior, development, and treatment response. This study investigated mesenchymal and stemness transdifferentiation of bladder cancer cell line due to environmental stress of K. pneumoniae. Cultures of urothelial bladder cancer cell line (T24) were infected with K. pneumoniae with different multiplicity of infection (MOI) for two and four days. Transdifferentiation-associated features were morphologically assessed. Moreover, transdifferentiation markers were estimated using Q-PCR and immunohistochemistry. Q-PCR data showed an increase in mesenchymal transdifferentiation traits; vimentin expression was upregulated, and cytokeratin19 expression downregulated significantly (P<0.001) compared with controls, which were emphasized by immunohistochemistry results. Moreover, stemness transdifferentiation markers expression increased significantly (P<0.001). The heterogeneous tumor cell population may be altered by bacterial infection, which improves cancer cells' migration and self-renewal ability. Thus, bacteria may be engaged in cancer progression and metastases.

Efficiency of Diode Laser as Root Canal Disinfectant Against Enterococcus faecalis: An in Vitro Study

The objective of the in vitro study is to determine the disinfection ability of diode laser 1064 nm at different powers and duration of times against E. faecalis and detect the optimal power and time of  diode laser 1064nm. Eighty human extracted single-rooted teeth were decoronated to a length 14mm from the apical foramen to the cervical  border of the root, then adjusted working length to 13 mm, autoclaved and inoculated with a suspension of E. faecalis at a concentration 4×105 cfu and  incubated  at 37˚C for 24 hours.  Sixty  samples were irradiated at  different powers and irradiation time 30 and 60 sec, the remainder 20 samples were control positive and negative groups.  All powers and times for laser 1064 nm had significant antimicrobial effects against E. faecalis. Diode laser device seems to be highly suitable for killing E. faecalis  if appropriate energy and irradiated time used

Transition of Care in Paediatric Surgery: Current practices and perspectives of paediatric surgeons in Malaysia

Objectives: Transition of care (TOC) from paediatric to adult care is still at an early stage in Malaysia. This study aimed to explore current practices and perspectives regarding TOC among paediatric surgeons in Malaysia. Methods: This study was carried out between June and December 2017. All 48 paediatric surgeons currently working in Malaysia were invited to participate in a questionnaire-based survey to assess demographic characteristics and practices and perspectives regarding TOC. Results: A total of 38 paediatric surgeons participated in the survey (response rate: 79.2%). Overall, 97.4% did not have an organised TOC model in their institution, with most (65.8%) caring for paediatric patients with complex surgical conditions until adulthood. Although the majority (86.8%) felt that care should be transitioned to adult surgeons with appropriate credentials, most surgeons (84.2%) nevertheless preferred to be involved in the management of adolescent patients after transition. However, there was no consensus regarding the most suitable age to begin the transition. Years of experience as a paediatric surgeon and place of practice did not affect overall TOC practice scores (P >0.050 each). The presence of adult comorbidities was considered the most common reason to initiate TOC (81.6%), while the lack of TOC guidelines was perceived to be the greatest barrier (84.2%). Conclusion: This study provides a better understanding of TOC from the point of view of paediatric surgeons in Malaysia. However, further studies involving other stakeholders (i.e. patients and adult surgeons) are needed to help formulate a suitable and successful TOC model in this setting.Keywords: Transition to Adult Care; Pediatrics; Adolescents; Surgery; Attitudes; Professional Practice; Malaysia

Optimizing the rheological properties of silica nano-modified bentonite mud using overlaid contour plot and estimation of maximum or upper shear stress limit.

An optimization based statistical (response surface) approach was used to evaluate the rheological properties of bentonite mud treated with silica nanoparticles. The overlaid contour plot established the feasible region for the various factor settings from multiple regression equations. The steepest method was used to further determine the optimal factor settings for minimum rheological properties and this was established at 6.3 wt.% bentonite content and 0.94 wt.% silica nanoparticles. The rheological properties of the bentonite mud containing and without silica nanoparticles was evaluated using a Hyperbolic (new) model and related with other oil industry based models: Herschel Bulkley, Sisko, Casson. The hyperbolic rheological model estimated the rheological behaviour of the nano-modified mud satisfactorily while also predicting a shear stress limit for the nano-modified mud. The maximum shear stress limit values for 6.3, 13 and 15 wt.% mud were 14.59, 61.74 and 107.4 Pa respectively. Upper shear stress values obtained from a 1.5 wt.% silica nanoparticle modified 6.3, 13 and 15 wt.% bentonite mud were 22.27, 72.62 and 171.3 Pa respectively, which represents an increment of 34.5 to 37.4% in the upper limit of shear stress. The effect of silica nanoparticles on the upper shear stress limit was quantified using a response surface design

Xeno-free trans-differentiation of adipose tissue-derived mesenchymal stem cells into glial and neuronal cells.

Mesenchymal stem cells (MSCs) are undifferentiated cells that have the ability of self-renewal and trans-differentiation into other cell types. They hold out hope for finding a cure for many diseases. Nevertheless, there are still some obstacles that limit their clinical transplantation. One of these obstacles are the xenogeneic substances added in either proliferation or differentiation media with subsequent immunogenic and infectious transmission problems. In this study, we aimed to replace fetal bovine serum (FBS), the main nutrient source for MSC proliferation with xeno-free blood derivatives. We tested the effect of human activated pure platelet-rich plasma (P-PRP) and advanced platelet-rich fibrin (A-PRF) on the proliferation of human adipose derived-MSCs (AD-MSCs) at different concentrations. For the induction of MSC neural differentiation, we used human cerebrospinal fluid (CSF) at different concentrations in combination with P-PRP to effect xeno-free/species-specific neuronal/glial differentiation and we found that media with 10% CSF and 10% PRP promoted glial differentiation, while media with only 10% PRP induced a neuron-like phenotype

Performance evaluation of solar-powered atmospheric water harvesting using different glazing materials in the tropical built environment: an experimental study

Water scarcity is a global issue, and its severity is expected to worsen in the near future, prompting further efforts to find new sources of freshwater. Solar-Powered Atmospheric Water Harvesting (SPAWH) is a promising passive approach for atmospheric water generation. This study aims to examine the thermal performance of different glazing materials and water production in SPAWH. The research consists of two phases: a laboratory test of various glazing materials and an experimental study to assess system efficiency in producing water in the tropics. The preliminary results indicated that glass demonstrated better thermal performance than acrylic in the lab, with higher thermal conductivity and less heat loss. The experimental findings showed that the maximum water produced by the proposed SPAWH (60 cm length, 60 cm width and 30 cm height) placed on a 30° tilt angle using glass (3 mm) and acrylic (3 mm) was 0.61 L/m2/day and 0.44 L/m2/day, respectively. The cost analysis revealed that produced water costs $0.18/kg for glass and$0.40/kg for acrylic, respectively. Atmospheric water could be harvested using SPAWH in the tropics, which would help to provide new opportunities for sustainable water supplies and development in these regions