Recombinant flounder growth hormone was overproduced in E. coli by using codon optimized synthetic gene and optimized expression conditions for high level production. The gene was cloned into PET-28a expression vector and transformed into E. coli BL21 (DE3). Induction at lower temperature, lower IPTG concentrations and richer growth media during expression resulted in increased expression level. The protein expression profile was analyzed by SDS-PAGE, the authenticity was confirmed by western blotting and the concentration was determined by Bradford assay. In addition, several attempts were made to produce soluble product and all resulted in insoluble product. The overexpressed protein was efficiently purified from inclusion bodies by moderate speed centrifugation after cell lysis. Among the solubilization buffers examined, buffer with 1% N-lauroylsarcosine in the presence of reducing agent DTT at alkaline pH resulted in efficient solubilization and recovery. The denaturant was removed by filtration and dialysis. The amount of the growth hormone recovered was significantly higher than previous reports that expressed native growth hormone genes in E. coli. The methodology adapted in this study, can be used to produce flounder growth hormone at large scale level so that it can be used in aquaculture. This approach may also apply to other proteins if high level expression and efficient purification is sought in E. coli.

Fungal and bacterial pathogens infect a diverse range of hosts including various plant and animal species. Fungal and bacterial diseases, especially of plants and aquatic animals, such as fish, lead to significant damage to crops and aquaculture, respectively, worldwide. The present study was conducted to isolate and characterize potent Bacillus strains with significant antagonistic activity against the major plant and fish pathogenic fungi and bacteria. We randomly collected 22 isolates of Bacillus from the soil, rhizosphere, and sediment from different parts of Bangladesh. Initial characterization, based on in vitro antagonistic activity on the culture plate, resulted in the selection of four gram-positive Bacillus sp. isolates. Among these, the isolate BC01, obtained from soil demonstrated the highest broad-spectrum anti-bacterial and anti-fungal activities. We confirmed the genus of BC01 to be Bacillus by morphological and biochemical tests as well as using molecular data analysis tools, including the study of 16s rDNA, phylogenetic relationship, and evolutionary divergence scores. The isolate significantly inhibited the mycelial growth of the plant pathogen, Penicillium digitatum and fish pathogen, Aphanomyces invadans in vitro. The anti-bacterial effect of the isolate was also evaluated against Pseudomonas spp. and Xanthomonas spp., the two deadliest plant pathogens, and Aeromonas veronii, Pseudomonas fluorescens, and Streptococcus iniae, three major fish pathogens that are primarily responsible for global aquaculture loss. The results of the present study could pave the way for developing potent drugs to combat microbial infection of plants and fish.

Methicillin-resistant Staphylococcus aureus (MRSA) has long been a common pathogen in healthcare facilities, but now, it has emerged as a problematic pathogen in the community setting as well. This study reported source, diagnosis and treatment of HA-MRSA and CA-MRSA. A total of sixty-five clinical samples (urine, pus, wound swab) were collected from clinical origin of Dhaka city, Bangladesh. All the isolates were tested phenotypically by conventional methods and genotypically by PCR targeting nuc, pvl and mecA genes. Finally sequencing was carried out for pvl gene to know the mutagenic variation or any amino acid changes in pvl gene. Chi square test was employed for statistical analysis. Patients of age group 51-60 years are more susceptible (46.15%) to MRSA, CA-MRSA or HA-MRSA infection. Female are (32.30%) more susceptible to MRSA infection. Among 65 isolates 53 isolates identified phenotypically as S. aureus. These were positive for amplification of nuc (270 bp) gene of S. aureus. Moreover, among 53 isolates 33 phenotypically considered as MRSA and 38 (72%) showed positive amplification for mecA (162 bp) gene. Among 38 MRSA isolates 22 (57.89%) confirmed as CA-MRSA and 16 (42.10%) as HA-MRSA. Finally, sequence analysis for lukS/F-PV genes from 4 representative isolates detected a new single nucleotide polymorphism in comparison with the control sequence. However, no amino acid changes were found. Statistical analysis showed HA-MRSA isolates were more commonly found in urine sample and CA-MRSA in pus and wound swab. CA-MRSA isolates were more resistant to tested antibiotics than HA-MRSA.

Mass propagation of date palm through indirect somatic embryogenesis or organogenesis has attracted the interest of commercial producers. But, this technique still faces some problems that hindered the production of date palm plantlets in vitro. Tissue browning is one of the serious problems that reduce callus growth and shoot regeneration. So the objective of the present study is to investigate the effect of cold pretreatment on callus growth, shoot regeneration, and polyphenol oxidase (PPO) activity during the callus culture. Results showed that a high survival rate of callus cultures (100%) were obtained when cultures were incubated in low temperature (cold treatment) for 45 and 75 days. On the other hand, total amount on phenolic compounds was also reduced to 0.47 and 0.53 mg GAE/g after same period of incubation (45 and 75 days respectively) at low temperature. In additional, our results showed that the highest frequency of shoot formation (66.67 and 73.34, %) and the highest shoot numbers (7.8 and 8.6 shoots/100 mg) were obtained from callus treated with low temperature for 45 and 75 days, respectively.

Ledebouria revoluta are important ethnomedicinal plant found in India and South Africa. Micropropagation via indirect shoot organogenesis had been established from three types of explant (i.e. scale leaf, leaf lamina and root) of L. revoluta. Scale leaf was found superior as compared to leaf lamina and root explant with respect to their organogenic callus induction potentiality. Murashige and Skoog (1962) [MS] media supplemented with 3.0 mg L-1 2,4-dichlorophenoxyacetic acid, 0.75 mg L-1 β-naphthoxyacetic acid were best effective for inducing organogenic callus. Maximum 17.0 ± 0.52 bulblets were induced from about 500 mg of callus within 42-46 days sub-culturing on a medium containing 0.75 mg L-1 kinetin. The bulblets were matured (86.7% success) after one month culture on the same medium composition. The best result of in vitro root induction with 100% response and 8.4 ± 0.31 roots per bulb was achieved after 18 days of implantation on MS medium containing 2.0 mg L-1 indole-3-butyric acid. Plantlets were acclimatized with a 96.0% survival rate. Chromosomal studies revealed cytological stability of callus cells and all regenerants containing 2n = 30 chromosomes, same as parental plants. Antimicrobial activity of L. revoluta was tested against two Gram-positive bacteria, three Gram-negative bacteria and two fungi. The methanol and ethanol extract proved more effective against bacteria, whereas acetone and chloroform extract shows potential anti-fungal activities. Present protocol can be applied reliably to produce uniform planting materials in large scale. In addition, this efficient indirect regeneration pathway via callus culture opens a way for improvement through genetic transformation.

The effect of some factors on in vitro consecutive micropropagation behavior of Antigonon leptopus was examined including those of culture establishment, shootlets multiplication, rooting and acclimatization stages. The highest percent of aseptic cultures and survival of explants (100%) were obtained as a result of using Clorox 10% for 3 min followed by MC 0.1% for 2 min while, using each of them individually (Clorox 20% or MC 0.1%) for 5 min caused the highest percent of shoot formation. During the multiplication stage, the highest percent of shoot formation was reached to 100% with repeating culture of explants (two times) on MS medium supplemented with 2ip at 1.0 and IBA at 0.2 mg/l. The highest numbers of shootlets/explant were obtained when 2.0 mg/l of BAP or 0.5 mg/l BA + 0.2 mg/l of IBA were added to MS culture medium. Culturing the explants on MS medium supplemented with 2ip at 0.5 or 1.0 mg/l each combined with 0.2 mg/l of IBA showed the longest shootlets. Reducing the strength of culture media to ½ or ¾ had promotion effect on rooting formation of shootlets. The best results of plant acclimatization (survival percent, plant height and root length) were obtained by using sand or peat moss soil. The amplified DNA fragments using B7, B9 and C19 primers for mother and micropropagated plants showed that the produced pattern by primer B7 had a maximum number of 10 bands of DNA fragments with molecular size ranging between 1025.57 and 176.36 bp, micropropagated plants showed 95.2% similarity in relation to mother plant.

Triterpenoid saponins are a diverse group of bioactive compounds, which are used for possessing of many biomedical and pharmaceutical products. Generally, squalene synthase (SQS) is defined as an emerging and essential branch point enzyme far from the major pathway of isoprenoids biosynthetic and a latent adjusting point, which manages carbon flux into triterpenes biosynthesis and sterols. The present study deals with the detailed characterization of SQS by bioinformatics approaches to evaluate physicochemical properties, structural characteristics including secondary and 3D structure prediction and functional analysis from eight plants related to Fabaceae family and Arabidopsis thaliana. Bioinformatics analysis revealed that SQS proteins have two transmembrane regions in the C-terminal. The predicted motifs were used to design universal degenerate primers for PCR analysis and other molecular applications. Phylogenetic analysis showed conserved regions at different stretches with maximum homology in amino acid residues within all SQSs. The secondary structure prediction results showed that the amino acid sequence of all squalene synthases had α helix and random coil as the main components. The reliability of the received model was confirmed using the ProSA and RAMPAGE programs. Determining of active site by CASTp proposes the possibility of using this protein as probable medication target. The findings of the present study may be useful for further assessments on characterization and cloning of squalene synthase.

Mesenchymal stromal cells (MSCs) can be easily isolated form craniofacial bones during routine dentistry procedures. Due to their embryological origin from neural crest, they represent a suitable cell population to study cell-biomaterial interaction in the craniofacial field, including osteoinductive/osteointegrative processes. The biological and immunomodulatory properties of MSCs may be influenced by chemistry and topography of implant surfaces. We investigated if and how three different titanium surfaces, machined (MCH), sandblasted with resorbable blasting medium (RBM), and Ca++-nanostructured (NCA), may affect biological activity, osseointegration, and immunomodulatory properties of craniofacial MSCs. Cell proliferation, morphology, osteogenic markers, and FasL were evaluated on MSCs isolated from the mandibular bone after seeding on these three different surfaces. No statistically significant differences in cell proliferation were observed whereas different morphologies and growth patterns were detected for each type of surface. No difference in the expression of osteogenic markers was revealed. Interestingly, FasL expression, involved in the immunomodulatory activity of stem cells, was influenced by surface properties. Particularly, immunofluorescence analysis indicated that FasL expression increased on MCH surface compared to the others confirming the suggested role of FasL in promoting osteogenic differentiation. Titanium surface treatments and topography might reflect different biological behaviours of craniofacial MSCs and influence their osseointegration/immunomodulation properties.

Trees of the Copaifera genus are native to the tropical regions of Latin America and Western Africa. Copaifera sp is widely used as a popular medicine and it has various ethnopharmacological indications, including gonorrhea, bronchitis, asthma, skin ulcers, ulcers, sore throat, uterine infections, general inflammations, cancer, and leishmanioses. Kaurenoic acid is a naturally occurring diterpene found in Copaifera and has been used as an anti-inflammatory, treatment of ulcer, leishmaniasis, and cancer. Bearing in mind the fact that the Ames test is an excellent tool to assess the safety of extracts, oils, and phytochemicals isolated from medicinal plants, from it, we evaluate the mutagenic potential of four species, between oleoresins (C. oblongifolia; C. langsdorffii) and leaves extracts (C. lucens; C. multijuga), of the Copaifera genus and also of kaurenoic acid, which is one of its major compounds. The results showed that the Copaifera spp. and kaurenoic acid did not induce an increase in the number of revertant colonies, without mutagenic effect in experiments, in the all concentrations evaluated by Ames test. The results obtained in our study support the safe use of the Copaifera genus medicinal plants selected and of kaurenoic acid.

Pentraxin 3 (PTX3), an acute-phase inflammation protein produced by several cell types, has long been described as a possible biomarker for age-related cardiovascular and cerebrovascular diseases. Although several mechanisms of action have been identified to date in the vascular and immune systems, the direct effects of PTX3 on isolated endothelial cells at morphological and metabolic levels remain unknown.

Nowadays, very few patients with non-variceal upper gastrointestinal bleeding fail endoscopic hemostasis (refractory NVUGIB). This subset of patients poses a clinical dilemma: should they be operated on or referred to transcatheter arterial embolization (TAE)?

The purpose of an experimental design is to improve the productivity of experimentation. It is an efficient procedure for planning experiments, so the data obtained can be analyzed to yield a valid and objective conclusion. This approach has been used as an important tool in the optimization of different analytical approaches. A D-optimal experimental design was used here, for the first time, to optimize the experimental conditions for the detection of reactive oxygen species (ROS) produced by human blood from healthy donors, a biological matrix that better resembles the physiologic environment, following stimulation by a potent inflammatory mediator, phorbol-12-myristate-13-acetate (PMA). For that purpose, different fluorescent probes were used, as 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), 2-[6-(4'-amino)-phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF), and 10-acetyl-3,7-dihydroxyphenoxazine (amplex red). The variables tested were the human blood dilution, and the fluorescent probe and PMA concentrations. The experiments were evaluated using the Response Surface Methodology and the method was validated using specific compounds. This model allowed the search for optimal conditions for a set of responses simultaneously, enabling, from a small number of experiments, the evaluation of the interaction between the variables under study. Moreover, a cellular model was implemented and optimized to detect the production of ROS using a yet nonexplored matrix, which is human blood.

Surgical training is an increasingly controversial topic. Concerns have been raised about both training opportunities becoming scarcer and poorer outcomes in operations led by surgical trainees; despite the evidence base for this being mixed. This retrospective cohort study aims to compare outcomes following carotid endarterectomy in patients who were operated on by a surgical trainee to those operated on by consultants.

Although there is an increasing interest for bioactive food protein hydrolysates as valuable ingredients for functional food and dietary supplement formulations, their potential applications are hampered by their insufficient stability in physiological conditions. In this study, an innovative strategy based on nanomaterials was developed in order to increase the hempseed hydrolysate stability and the anti-diabetic properties, through their encapsulation into ionic self-complementary RADA16 peptide based-hydrogels. Atomic force microscope (AFM) morphological analysis indicated that the new nanomaterials were composed of a nanofibril network, whose increased diameter in respect to native RADA16 suggests the presence of transient non-covalent interactions among the RADA16 supramolecular assemblies and the embedded hempseed peptides. Structural analysis by FT-IR spectroscopy indicated typical β-sheet signatures. The RADA16-hempseed protein hydrolysate hydrogel was shown to act as a novel dipeptidyl peptidase IV (DPPIV) inhibitor in different biological assays. Finally, this nanoformulation was used as a drug delivery system of the anti-diabetic drug sitagliptin, helping to reduce its dosage and eventually associated side-effects.

Salivary alpha-amylase (sAA) and salivary immunoglobulin A (sIgA) have been proposed as biomarkers for research on the mucosal immune system and on stress. Expression of both sAA and sIgA has been described to follow opposing diurnal patterns. This knowledge is crucial for the interpretation of studies using these biomarkers.

Avoiding intubation and promoting noninvasive modes of ventilator support including continuous positive airway pressure (CPAP) in preterm infants minimizes lung injury and optimizes neonatal outcomes. Discharge home on oxygen is an expensive morbidity in very preterm infants (VPI) with lung disease. In 2007 a standardized bundle was introduced for VPI admitted to the neonatal care unit (NICU) which included delayed cord clamping (DCC) at birth and noninvasive ventilation as first-line cardiorespiratory support in the delivery room (DR), followed by bubble CPAP upon NICU admission.

To retrospectively evaluate the impact of comorbidities on treatment choice, 12-month clinical response, and 24-month retention rate in a cohort of patients with rheumatoid arthritis (RA) treated with a first-line tumor necrosis factor alpha inhibitor (TNFi), by using for the first time the Rheumatic Disease Comorbidity Index (RDCI).

The purpose of this study was to assess the diagnostic value of arginine-glycine-aspartic acid (RGD) PET/CT for tumor detection in patients with suspected malignant lesions and to determine the predictive performance of RGD PET/CT in identifying responders. Methods. The PubMed (Medline), EMBASE, Cochrane Library, and Web of Science databases were systematically searched for potentially relevant publications (last updated on July 28th, 2018) reporting the performance of RGD PET in the field of oncology. Pooled sensitivities, specificities, and diagnostic odds ratios (DORs) were calculated for parameters. The areas under the curve (AUCs) and Q⁎ index scores were determined from the constructed summary receiver operating characteristic (SROC) curve. We explored heterogeneity by metaregression. Results. Nine studies, five including 216 patients that determined diagnostic performance and three including 75 patients that determined the predictive value of parameters, met our inclusion criteria. The pooled sensitivity, pooled specificity, DOR, AUC, and Q⁎ index score of RGD PET/CT for the detection of underlying malignancy were 0.85 (0.79-0.89), 0.93 (0.90-0.96), 48.35 (18.95-123.33), 0.9262 (standard error=0.0216), and 0.8606 for SUVmax and 0.86 (0.80-0.91), 0.92 (0.88-0.94), 40.49 (14.16-115.77), 0.9312 (SE=0.0177), and 0.8665 for SUVmean, respectively. The pooled sensitivity, pooled specificity, DOR, AUC, and Q⁎ index score of RGD PET/CT for identifying responders were 0.80 (0.59-0.93), 0.74 (0.60-0.85), 15.76 (4.33-57.32), 0.8682 (0.0539), and 0.7988, respectively, for SUVmax at baseline. Conclusion. The interesting but preliminary data in this meta-analysis demonstrate that RGD PET/CT may be an ideal diagnostic tool for detecting underlying malignancies in patients suspected of having tumors and may be able to efficiently predict short-term outcomes.

We report an extraordinary case of a woman, exposed to diethylstilbestrol in utero, who developed clear cell adenocarcinoma of the cervix with a concurrent polymerase-Ɛ (POLE) somatic mutation. The tumor exhibited the classic phenotypic characteristics of POLE-mutated tumors originating from other organs (e.g. the uterus or the colon) including increased tumor infiltrating lymphocytes and high PD-L1 expression and has remained in remission since completion of primary therapy for >4 years. This case highlights the importance of next generation sequencing in unraveling the biology of rare tumors and supports that the presence of a POLE mutation and the associated ultramutated state confers a unique phenotype of higher immunogenicity and possibly improved prognosis in a tissue-agnostic manner, i.e. regardless of the type of cancer where the POLE mutation is present.

Neoadjuvant chemotherapy or chemoradiotherapy is used widely before tumour resection in cancer of the gastro-oesophageal junction (GOJ). Strategies to improve treatment tolerability are warranted. This study examined the safety and feasibility of preoperative exercise training during neoadjuvant treatment in these patients.