<Emphasis Type="Italic">In silico</Emphasis> panning for a non-competitive peptide inhibitor

Background  

Peptide ligands have tremendous therapeutic potential as efficacious drugs. Currently, more than 40 peptides are available in the market for a drug. However, since costly and time-consuming synthesis procedures represent a problem for high-throughput screening, novel procedures to reduce the time and labor involved in screening peptide ligands are required. We propose the novel approach of 'in silico panning' which consists of a two-stage screening, involving affinity selection by docking simulation and evolution of the peptide ligand using genetic algorithms (GAs). In silico panning was successfully applied to the selection of peptide inhibitor for water-soluble quinoprotein glucose dehydrogenase (PQQGDH).     

Selection of Anti-Sulfadimidine Specific ScFvs from a Hybridoma Cell by Eukaryotic Ribosome Display

Background

Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes.

Methodology/Principal Findings

In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM₂) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA–ribosome–antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM₂-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM₂ by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis.

Conclusions/Significance

The selection of anti-SM₂ specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs.     

Isolating substrates for an engineered alpha-lytic protease by phage display

Panning of a substrate phage library with an -lytic protease mutant showed that substrate phage display can be used to isolate sequences with improved protease sensitivity even for proteases of relatively broad specificity. Two panning experiments were performed with an engineered -lytic protease mutant known to have a preference for cleavage after His or Met residues. Both experiments led to the isolation of protease-sensitive phage containing linker sequences in which His and Met residues were enriched compared with the initial library. Despite the relatively hydrophobic substrate binding site of the enzyme, the predominant protease-sensitive sequence isolated from the second library panning had the sequence Asp-Ser-Thr-Met. Kinetic studies showed that this sequence was cleaved up to 4.5-fold faster than rationally designed positive controls. Protease-resistant phage particles were also selected and characterized, with the finding that Gly and Pro appeared frequently at the putative P₄ positions, whereas Asp dominated the putative P₁ position.     

Bridging the phenotypic gap: Real-time assessment of mitochondrial function and metabolism of the nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

The ATP levels of an organism are an important physiological parameter that is affected by genetic make up, ageing, stress and disease.     

A laminar flow model of aerosol survival of epidemic and non-epidemic strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolated from people with cystic fibrosis

Background  

Cystic fibrosis (CF) is an inherited multi-system disorder characterised by chronic airway infection with pathogens such as Pseudomonas aeruginosa.     

Albumin domain II mutant with high bilirubin binding affinity has a great potential as serum bilirubin excretion enhancer for hyperbilirubinemia treatment

Background

4Z,15Z-bilirubin-IXα (BR), an endogenous toxic compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. Our previous findings suggest that both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of BR, and especially Lys199 in stand-alone domain II plays a prominent role in the renal elimination of BR. Our hypothesis is that HSA-domain II with high BR binding would be a useful therapeutic agent to treat hyperbilirubinemia in patients with impaired liver function.

Methods

Unbound BR concentrations were determined using a modified HRP assay. To evaluate the effect of pan3_3-13 domain II mutant in promoting urinary BR excretion, the serum concentration and urinary excretion amount of BR were determined using bile duct ligation mice.

Results

After three or six rounds of panning, pan3_3-13 and pan6_4 were found to have a significantly higher affinity for BR than wild-type domain II. Administration of pan3_3-13 significantly reduced serum BR level and increased its urinary excretion in the disease model mice as compared to wild-type domain II treatment.

Conclusions

These results suggest that pan3_3-13 has great potential as a therapeutic agent that promotes urinary BR excretion in hyperbilirubinemia.

General significance

This is the first study to be applied to other HSA bound toxic compounds that are responsible for the progression of disease, thereby paving the way for the development of non-invasive and cost effective blood purification treatment methods.     

SurfaceSlide: a multitouch digital pathology platform

Background

Digital pathology provides a digital environment for the management and interpretation of pathological images and associated data. It is becoming increasing popular to use modern computer based tools and applications in pathological education, tissue based research and clinical diagnosis. Uptake of this new technology is stymied by its single user orientation and its prerequisite and cumbersome combination of mouse and keyboard for navigation and annotation.

Methodology

In this study we developed SurfaceSlide, a dedicated viewing platform which enables the navigation and annotation of gigapixel digitised pathological images using fingertip touch. SurfaceSlide was developed using the Microsoft Surface, a 30 inch multitouch tabletop computing platform. SurfaceSlide users can perform direct panning and zooming operations on digitised slide images. These images are downloaded onto the Microsoft Surface platform from a remote server on-demand. Users can also draw annotations and key in texts using an on-screen virtual keyboard. We also developed a smart caching protocol which caches the surrounding regions of a field of view in multi-resolutions thus providing a smooth and vivid user experience and reducing the delay for image downloading from the internet. We compared the usability of SurfaceSlide against Aperio ImageScope and PathXL online viewer.

Conclusion

SurfaceSlide is intuitive, fast and easy to use. SurfaceSlide represents the most direct, effective and intimate human–digital slide interaction experience. It is expected that SurfaceSlide will significantly enhance digital pathology tools and applications in education and clinical practice.     

Time-dependent structural transformation analysis to high-level Petri net model with active state transition diagram

Background  

With an accumulation of in silico data obtained by simulating large-scale biological networks, a new interest of research is emerging for elucidating how living organism functions over time in cells.     

Atopic dermatitis‐mitigating effects of new Lactobacillus strain,Lactobacillus sakei probio 65 isolated from Kimchi

Aims

Atopic dermatitis (AD) is an inflammatory skin disease. Probiotics have been reported to modulate immune responses and thus are now being suggested as potential treatments for allergies. In this study, we investigated the inhibitory effects of Lactobacillus sakei probio 65 isolated from Kimchi on artificially inducing AD in NC/Nga mice.

Methods and Results

Oral administration of viable or heat‐inactivated Lact. sakei probio 65 improved the condition of skin and reduced scratching frequency. Serum levels of IgE and cutaneous T‐cell‐attracting chemokine (CTACK) were significantly decreased by this therapy. Dead Lact. sakei probio 65 also decreased IL‐4 and IL‐6 serum concentrations. Moreover, both live and dead Lact. sakei probio 65 inhibited the expression of Thymus and activation‐regulated chemokine and CTACK in AD‐like skin lesions. The increased levels of Foxp3 expression in the lesional skin and ears were also suppressed by Lact. sakei probio 65. In addition, Lact. sakei probio 65 inhibited β‐hexosaminidase release and the secretion of IL‐4, TNF‐α and IL‐6 from RBL‐2H3 cells.

Conclusions

Oral treatment with both viable and heat‐inactivated Lact. sakei probio 65 inhibits skin inflammation and AD‐like skin lesions, as well as mast cell activation.

Significance and Impact of the Study

Lactobacillus sakei probio 65 has an inhibitory effect on atopic dermatitis‐like skin lesions and may represent an effective new anti‐inflammatory agent.     

The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies

Background  

Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.     

Effect of menstrual cycle phase and hormonal treatments on evaluation of tubal patency in baboons

Background

We evaluated whether menstrual cycle phase influences the assessment of tubal patency by hysterosalpingography (HSG) in baboons.

Methods

Retrospective analysis of baseline tubal patency studies and serum estradiol (E₂) and progesterone (P4) values obtained from female baboons used as models for development of non‐surgical permanent contraception in women. The main outcome measure was bilateral tubal patency (BTP) in relationship with estradiol level.

Results

Female baboons (n = 110) underwent a single (n = 81), two (n = 26), or three (n = 3) HSG examinations. In 33/142 (23%) HSG examinations, one or both tubes showed functional occlusion (FO). The median E₂ in studies with BTP (49 pg/mL) was significantly higher than in those studies with FO (32 pg/mL, P = .005). Among 18 animals with repeat examinations where serum E₂ changed from <60 to ≥ 60 pg/mL, 13 results changed from FO to BTP (P = .0001). No sets showed a change from BTP to FO with an increase in estradiol.

Conclusion

In baboons, functional occlusion of the fallopian tube is associated with low estradiol levels, supporting a role for estrogen‐mediated relaxation of the utero‐tubal junction.     

Human appropriation of net primary production as driver of change in landscape-scale vertebrate richness

Aim

Land use is the most pervasive driver of biodiversity loss. Predicting its impact on species richness (SR) is often based on indicators of habitat loss. However, the degradation of habitats, especially through land-use intensification, also affects species. Here, we evaluate whether an integrative metric of land-use intensity, the human appropriation of net primary production, is correlated with the decline of SR in used landscapes across the globe.

Location

Global.

Time period

Present.

Major taxa studied

Birds, mammals and amphibians.

Methods

Based on species range maps (spatial resolution: 20 km × 20 km) and an area-of-habitat approach, we calibrated a “species–energy model” by correlating the SR of three groups of vertebrates with net primary production and biogeographical covariables in “wilderness” areas (i.e., those where available energy is assumed to be still at pristine levels). We used this model to project the difference between pristine SR and the SR corresponding to the energy remaining in used landscapes (i.e., SR loss expected owing to human energy extraction outside wilderness areas). We validated the projected species loss by comparison with the realized and impending loss reconstructed from habitat conversion and documented by national Red Lists.

Results

Species–energy models largely explained landscape-scale variation of mapped SR in wilderness areas (adjusted R²-values: 0.79–0.93). Model-based projections of SR loss were lower, on average, than reconstructed and documented ones, but the spatial patterns were correlated significantly, with stronger correlation in mammals (Pearson's r = 0.68) than in amphibians (r = 0.60) and birds (r = 0.57).

Main conclusions

Our results suggest that the human appropriation of net primary production is a useful indicator of heterotrophic species loss in used landscapes, hence we recommend its inclusion in models based on species–area relationships to improve predictions of land-use-driven biodiversity loss.     

Anti-N-methyl-D-aspartate receptor encephalitis with serum anti-thyroid antibodies and IgM antibodies against Epstein-Barr virus viral capsid antigen: a case report and one year follow-up

Background  

Anti-N-methyl-D-aspartate receptor encephalitis is an increasingly common autoimmune disorder mediated by antibodies to certain subunit of the N-methyl-D-aspartate receptor. Recent literatures have described anti-thyroid and infectious serology in this encephalitis but without follow-up.     

Brief report: Lactobacillus bulgaricus GLB44 (Proviotic™) plus esomeprazole for Helicobacter pylori eradication: A pilot study

Background

Recent studies of Lactobacillus delbrueckii subsp. bulgaricus GLB44 plus a proton‐pump inhibitor (PPI) reported cures of more than 90% of patients with active Helicobacter pylori infections.

Aim

To confirm the high H. pylori cure rates reported previously.

Method

A pilot study was done in healthy H. pylori‐infected volunteers using 3‐gram sachet (3 billion cells) of L. delbrueckii GLB44 plus 22.3 mg of esomeprazole b.i.d., for 14 days. The result was determined by urea breath testing 4 weeks after therapy. Stopping rules required for ending enrollment if less than 3 of the first 10 subjects were cured.

Results

Nine subjects were entered and because all failed to achieve negative urea breath test, the stopping rule required the study to end.

Conclusion

We were unable to confirm reports of achieving a high H. pylori cure rate with L. delbrueckii GLB44 plus a PPI.     

An immune reaction may be necessary for cancer development

Background  

The hypothesis of immunosurveillance suggests that new neoplasms arise very frequently, but most are destroyed almost at their inception by an immune response. Its correctness has been debated for many years.     

Tooth loss and its relationship with protein intake by elderly Brazilians—A structural equation modelling approach

Objective

This study aimed at assessing the relationship between self‐perceived tooth loss and wearing dentures, on the one hand, and the consumption of protein, on the other hand, among the elderly population of Botucatu, SP. Food consumption tends to decrease with ageing, especially protein intake, and one of the causes could be the precariousness of oral health. Several risk factors associated with deficient dietary protein intake have been identified, namely greater physical dependence, reduced caloric intake and food insecurity, but no studies have analysed whether tooth loss and prostheses interfere with protein intake.

Methods

An interview was conducted among 365 elderly individuals, in which we examined oral health‐related quality of life (OHRQoL) as the only latent variable, in a 24‐hour nutritional assessment dietary recall repeated 3 times, conducted in person by a trained nutritionist and also performed an analysis of nutritional needs using the Nutrition Data System Research (NDSR) Program.

Results

The structural equation model, performed using Stata v.14, showed that lack of teeth (standardised coefficient [SC] = 0.21, P < .001), and prosthesis use (SC = ?0.21, P < .001) was associated with OHRQoL. Lack of teeth had a direct effect on the consumption of animal protein (SC = 0.08, P = .02), a strong total effect on animal protein intake (SC = 0.51, P = .04) and a medium effect on total protein intake (SC = 0.20, P = .03), adjusted for confounders (depression and medical problems).

Conclusion

Tooth loss had a strong and significant total effect on animal protein intake and a medium effect on total protein intake among elderly Brazilians.     

Intravenous administration of anti-vascular endothelial growth factor humanized monoclonal antibody bevacizumab improves articular cartilage repair

Introduction  

In this study, we investigate the efficacy of repairing an osteochondral defect in rabbit knee joints by administering bevacizumab, a humanized monoclonal anti-vascular endothelial growth factor (VEGF) antibody.     

An update on the mouse liver proteome

Background  

Decoding of the liver proteome is subject of intense research, but hampered by methodological constraints. We recently developed an improved protocol for studying rat liver proteins based on 2-DE-MALDI-TOF-MS peptide mass finger printing.     

Erythrocyte membrane fatty acid profile and saturation index in gallbladder carcinogenesis: a case-control study

Background  

Gallbladder cancer is a common neoplasm of biliary tract, with an unknown etiology.