In silico designing and characterization of outer membrane protein K (OmpK) from Vibrio anguillarum and its expression in Nicotiana tabacum for the development of a plant-based vaccine against fish vibriosis.

Vibriosis is caused by Vibrio anguillarum in various species of aquaculture. A novel, secure, and stable vaccine is needed to eradicate vibriosis. Here, for reverse vaccinology and plant-based expression, the outer membrane protein K (OmpK) of V. anguillarum was chosen due to its conserved nature in all Vibrio species. OmpK, an ideal vaccine candidate against vibriosis, demonstrated immunogenic, non-allergic, and non-toxic behavior by using various bioinformatics tools. Docking showed the interaction of the OmpK model with TLR-5. In comparison to costly platforms, plants can be used as alternative and economic bio-factories to produce vaccine antigens. We expressed OmpK antigen in Nicotiana tabacum using Agrobacterium-mediated transformation. The expression vector was constructed using Gateway® cloning. Transgene integration was verified by polymerase chain reaction (PCR), and the copy number via qRT-PCR, which showed two copies of transgenes. Western blotting detected monomeric form of OmpK protein. The total soluble protein (TSP) fraction of OmpK was equivalent to 0.38% as detected by ELISA. Mice and fish were immunized with plant-derived OmpK antigen, which showed a significantly high level of anti-OmpK antibodies. The present study is the first report of OmpK antigen expression in higher plants for the potential use as vaccine in aquaculture against vibriosis, which could provide protection against multiple Vibrio species due to the conserved nature OmpK antigen.

Monolithic 45 Degree Deflecting Mirror as a Key Element for Realization of 2D Arrays of Laser Diodes Based on AlInGaN Semiconductors.

In this study, we propose a solution for realization of surface emitting, 2D array of visible light laser diodes based on AlInGaN semiconductors. The presented system consists of a horizontal cavity lasing section adjoined with beam deflecting section in the form of 45° inclined planes. They are placed in the close vicinity of etched vertical cavity mirrors that are fabricated by Reactive Ion Beam Etching. The principle of operation of this device is confirmed experimentally; however, we observed an unexpected angular distribution of reflected rays for the angles lower than 45°, which we associate with the light diffraction and interference between the vertical and deflecting mirrors. The presented solution offers the maturity of edge-emitting laser technology combined with versatility of surface-emitting lasers, including on-wafer testing of emitters and addressability of single light sources.

InGaN Laser Diodes with Etched Facets for Photonic Integrated Circuit Applications.

The main objective of this work is to demonstrate and validate the feasibility of fabricating (Al, In) GaN laser diodes with etched facets. The facets are fabricated using a two-step dry and wet etching process: inductively coupled plasma-reactive ion etching in chlorine, followed by wet etching in tetramethylammonium hydroxide (TMAH). For the dry etching stage, an optimized procedure was used. For the wet etching step, the TMAH temperature was set to a constant value of 80 °C, and the only variable parameter was time. The time was divided into individual steps, each of 20 min. To validate the results, electro-optical parameters were measured after each step and compared with a cleaved reference, as well as with scanning electron microscope imaging of the front surface. It was determined that the optimal wet etching time was 40 min. For this time, the laser tested achieved a fully comparable threshold current (within 10%) with the cleaved reference. The described technology is an important step for the future manufacturing of photonic integrated circuits with laser diodes integrated on a chip and for ultra-short-cavity lasers.

Plastidial Expression of 3ß-Hydroxysteroid Dehydrogenase and Progesterone 5ß-Reductase Genes Confer Enhanced Salt Tolerance in Tobacco.

The short-chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and progesterone 5-ß-reductases (P5ßR1, P5ßR2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, their role in plant stress, especially in salinity stress management, remains unexplored. In the present study, transplastomic tobacco plants were developed by inserting the 3ß-HSD, P5ßR1 and P5ßR2 genes. The integration of transgenes in plastomes, copy number and transgene expression at transcript and protein level in transplastomic plants were confirmed by PCR, end-to-end PCR, qRT-PCR and Western blot analysis, respectively. Subcellular localization analysis showed that 3ß-HSD and P5ßR1 are cytoplasmic, and P5ßR2 is tonoplast-localized. Transplastomic lines showed enhanced growth in terms of biomass and chlorophyll content compared to wild type (WT) under 300 mM salt stress. Under salt stress, transplastomic lines remained greener without negative impact on shoot or root growth compared to the WT. The salt-tolerant transplastomic lines exhibited enhanced levels of a series of metabolites (sucrose, glutamate, glutamine and proline) under control and NaCl stress. Furthermore, a lower Na+/K+ ratio in transplastomic lines was also observed. The salt tolerance, mediated by plastidial expression of the 3ß-HSD, P5ßR1 and P5ßR2 genes, could be due to the involvement in the upregulation of nitrogen assimilation, osmolytes as well as lower Na+/K+ ratio. Taken together, the plastid-based expression of the SDR genes leading to enhanced salt tolerance, which opens a window for developing saline-tolerant plants via plastid genetic engineering.

Impact of COVID-19 on Pakistani dentists: a nationwide cross sectional study.

BACKGROUND: The COVID-19 outbreak which developed into a public health crisis has raised concerns regarding infection control among health care workers particularly dentists all over the world. The aim of this survey was to assess awareness, fear and compliance with practice modification according to CDC guidelines during COVID-19 pandemic among Pakistani dentists. METHODS: A cross-sectional study was conducted using an online survey questionnaire. The questionnaire was designed on Google Forms and was distributed among all seven regions of Pakistan through social media and WhatsApp after carrying out the reliability analysis. Statistical analysis was performed using SPSS 20.0. Question wise analysis using frequencies and percentages was done. Pearson correlation and Kruskal Wallis test was applied to check association of awareness level with qualification and workplace setting. RESULTS: A total of 313 dentists participated and submitted the form online from all regions of Pakistan. The response rate was quite satisfactory as Pakistan was under an official lockdown and most of the hospitals/clinics were either closed or operating with minimum staff. Most of the dentists were well aware of the CDC guidelines. However, 75% of the dentists were afraid of getting infected and 88% of them were anxious while providing treatment. Sixty-eight percent of them were avoiding aerosol generating procedures and only 28% were using rubber dam isolation. Regional analysis was also done and areas of poor compliance were identified. Only 38.5% dentists in Balochistan were using N95 masks and none of the dentists (0%) were using rubber dam isolation. A large number of dentists (80.9%) were afraid and wanted to close their dental practice in Khyber Pakhtunkhwa. Furthermore, a positive correlation was seen between the designation and awareness level (p = 0.01). Similarly, significant correlation (p = 0.03) was seen between qualification and workplace setting. CONCLUSION: The results of the study can help devise strategies to ensure adherence with infection control guidelines in regions with poor compliance. Initiation of awareness programs to help overcome fear and train the faculty and staff in the targeted areas would greatly contribute towards reducing the spread of infection and thus lowering the healthcare burden in a third world country like Pakistan.

Expression of ESAT-6 antigen from Mycobacterium tuberculosis in broccoli: An edible plant.

Tuberculosis (TB) is one of the major infectious diseases caused by Mycobacterium tuberculosis. The development of an effective and economical vaccine for controlling TB is essential especially for developing countries. Edible plants can serve as biofactories to produce vaccine antigens. In this study, 6 kDa early secretory antigenic target (ESAT-6) of M. tuberculosis was expressed in Brassica oleracea var. italica via Agrobacterium-mediated transformation to facilitate oral delivery of antigen. ESAT-6 gene was cloned using Gateway® cloning strategy. Transformation and presence of transgene was confirmed through PCR. Expression level of transgene was calculated via quantitative real-time PCR (qRT-PCR) and the maximum integrated transgene number was two. Maximum amount of total soluble fraction of ESAT-6 was evaluated by immunoblotting, estimated to accumulate up to 0.5% of total soluble protein. The recombinant ESAT-6 protein was further purified and detected using silver staining and Western blotting. ESAT-6 protein induced humoral immune response in mice immunized orally and subcutaneously. The expression of M. tuberculosis antigen in edible plants could aid in the development of cost-effective and oral delivery of an antigen-based subunit vaccine against TB. To the best our knowledge, it is the first report of expression of a vaccine antigen in broccoli.

Chloroplast-based inducible expression of ESAT-6 antigen for development of a plant-based vaccine against tuberculosis.

Mycobacterium tuberculosis causes tuberculosis in humans. The major disease burden of tuberculosis lies in developing countries. Lack of an effective vaccine for adults is one of the major hurdles for controlling this deadly disease. In the present study, 6 kDa early secretory antigenic target (ESAT-6) of M. tuberculosis was inducibly expressed in chloroplasts of Nicotiana tabacum. The expression of ESAT-6 in chloroplasts was controlled by T7 promoter that was activated by nuclear-generated signal peptide. Tobacco plants, containing nuclear component, were transformed via biolistic bombardment with pEXP-T7-ESAT-6 obtained by Gateway® cloning. Transformation and homoplasmic status of transplastomic plants was confirmed by polymerase chain reaction and Southern blotting. Plants were induced for protein expression by spraying with 5% ethanol for 1 day, 3 days, 7 days and 10 days. ESAT-6 protein was detected by immunoblot analysis and maximum protein was obtained for 10 days induced plants that was estimated to accumulate up to 1.2% of total soluble fraction of protein. Transplastomic plants showed completely normal morphology. Transplastomic and untransformed plants became slightly chlorotic upon prolonged exposure to ethanol until 10 days. Taken together, this data could help in the development of an antigen-based subunit vaccine against tuberculosis.

Interplay of N acetyl cysteine and melatonin in regulating oxidative stress-induced cardiac hypertrophic factors and microRNAs.

Early and specific diagnosis of oxidative stress linked diseases as cardiac heart diseases remains a major dilemma for researchers and clinicians. MicroRNAs may serve as a better tool for specific early diagnostics and propose their utilization in future molecular medicines. We aimed to measure the microRNAs expressions in oxidative stress linked cardiac hypertrophic condition induced through stimulants as Endothelin and Isoproterenol. Cardiac hypertrophic animal models were confirmed by BNP, GATA4 expression, histological assays, and increased cell surface area. High oxidative stress (ROS level) and decreased antioxidant activities were assessed in hypertrophied groups. Enhanced expression of miR-152, miR-212/132 while decreased miR-142-3p expression was observed in hypertrophic condition. Similar pattern of these microRNAs was detected in HL-1 cells treated with H2O2. Upon administration of antioxidants, the miRNAs expression pattern altered from that of the cardiac hypertrophied model. Present investigation suggests that oxidative stress generated during the cardiac pathology may directly or indirectly regulate anti-hypertrophy pathway elements through microRNAs including antioxidant enzymes, which need further investigation. The down-regulation of free radical scavengers make it easier for the oxidative stress to play a key role in disease progression.

Comparison Of Two Intracanal Medicaments In Resolution Of Apical Radiolucency.

BACKGROUND: As a gold standard, calcium hydroxide has always been used as a root canal medicament against root canal pathogens but its combination with chlorhexidine is not being employed in routine despite its documented efficacy and results. The objective of the study is to compare calcium hydroxide and combination of calcium hydroxide with 2% CHX on the basis of radiographic evidence of resolution of periapical radiolucency when they are used as an interappointment dressing /intracanal medicament for 14 days.. METHODS: A total of 60 patients of either gender was included in the study. The age range was 15-60 years with the radiographic evidence of radiolucency of 2-4 mm in size. Both maxillary and mandibular teeth till first molar were selected. Teeth with previous endodontic treatment mentally handicapped and terminally ill patients were excluded. Selected patients were placed randomly into Group 1 (calcium hydroxide alone) and in Group 2 (calcium hydroxide in combination with 2% chlorhexidine gel). Obturation and permanent filling was done at 14th day. The patient was recalled after 90 days of obturation and a radiograph was taken. Final outcome was measured at the end of 90thday of obturation. RESULTS: The mean of percentage reduction in size of apical radiolucency at 3 months was 91.03% for group 1 and 97.26% for group 2 with p-value 0.13 (not significant). The results suggested equivalent results after medication with 2%chlorhexidine with calcium hydroxide but 2% chlorhexidine still showed better results than the group without CHX in terms of healing. CONCLUSIONS: This study concluded that calcium hydroxide in combination with 2% chlorhexidene as intracanal medicament results in better resolution of periapical radiolucency in terms of percentage resolution of periapical radiolucency.

Comparison of frequency of pain in root canal treatment using sodium hypochlorite and chlorhexidine as root canal irrigants.

OBJECTIVE: To compare the frequency of pain in root canal treatment using sodium hypochlorite and chlorhexidine as root canal irrigants. METHODS: The cross-sectional study was carried out from January to June 2016 at the Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan, and comprised patients aged 18-40 years with irreversible pulpitis involving a posterior tooth. The patients were divided into two equal groups, with Group-I receiving 2% chlorhexidine, and Group-II receiving 5.25% sodium hypochlorite. Following the use of the irrigants, the selected teeth were evaluated for pain at 72 hours using Visual Analogue Scale. RESULTS: There were 60 patients divided into groups of 30(50%) each. The overall mean age was 27.97±5.9 years. There were 32 (53.3%) females and 28 (46.6%) males. The mean post-operative pain score was 1.70±1.9 in Group-I and 1.90 ± 2.3 in Group-II (p=0.5). In Group-I, 25(83.3%) patients had no pain while in Group-II, 20(66.7%) had no pain (p=0.1). CONCLUSIONS: There was no difference in post-operative pain at 72 hours between 2% chlorhexidine and 5.25% sodium hypochlorite when used as irrigants.

Comparison of frequency of post-obturation pain of single versus multiple visit root canal treatment of necrotic teeth with infected root canals. A Randomized Controlled Trial.

OBJECTIVE: To determine the pain of single versus multiple visit endodontic treatment after obturation in teeth with necrotic pulps and infected canals. METHODS: The randomized controlled study was conducted from January to June 2016 at Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan. Dental patients aged 18-60 years of either gender were selected who were subjected to computer-generated randomisation. Both maxillary and mandibular single rooted teeth till second premolar were included. Single visit endodontic treatment to Group-I subjects was carried out by gaining access to canals, cleaning, shaping and obturation at same visit whereas in Group-II patients, who underwent multiple visits, cleaning and shaping was followed by placement of intra-canal medicament, while obturation was done 5 days later. Patients were recalled within 48 hours after obturation to record the pain score using visual analogue scale.. RESULTS: Of the 60 patients, 30(50%) were in each of the two groups. There was no significant difference in post-operative pain in the two groups (p=0.8). The frequency of post-operative pain was not significant either (p>0.05). The post-operative mean pain score was 2.23±1.736 in Group-I and 2.38±1.94 in Group-II (p=0.8).. CONCLUSIONS: Treatment carried out in single visit or multiple visits showed no difference in pain frequency.

Comparison Of Frequency Of Post Operative Sensitivity In Amalgam Restorations Using Copal Varnish And Dentin Adhesive Liner.

BACKGROUND: Micro leakage around the margins of a restoration is believed to be one of the main causes of postoperative sensitivity. Dental amalgam is a non-insulating material and has the potential to transfer heat and cold causing irritation of the pulp. Different dentin tubule sealers are used under amalgam restoration to compensate for this post-operative sensitivity. This study was conducted to compare the frequency of sensitivity in amalgam restorations using copal varnish and dentin adhesive liner (dentin bonding agent). METHODS: A total of 60 patients of either gender, aged 18-40 years having class 1 carries in posterior teeth were included. Teeth with restorations, dentinal sensitivity and patients taking analgesic drugs for chronic pain conditions were excluded. The selected patients were placed randomly into Group A (copal varnish) & Group B (dentin adhesive liner), by using computer generated table of random numbers. Restored teeth were evaluated 1-month post operatively for sensitivity. RESULTS: Mean age was 25.63±5.42 years. Out of 60 patients, 68.0% were females and 32.0% were males with a female to male ratio of 2:1. The mean post-operative pain score was 2.83±2.79 in Group A and in Group B, it was 1.43±2.14 with a p-value of 0.03. There was no pain on application of a cold stimulus in 14 (46.7%) patients in Group A (copal varnish) while in Group B (Dentin adhesive), no pain was seen in 23 (76.7%) patients with p-value of 0.02. CONCLUSIONS: This study concluded that dentin adhesive liner (dentin bonding agent) is better than copal varnish in reducing postoperative sensitivity in amalgam restorations.