A new candidate epitope-based vaccine against PspA PhtD of Streptococcus pneumoniae: a computational experimental approach.

Introduction: Pneumococcus is an important respiratory pathogen that is associated with high rates of death in newborn children and the elderly. Given the disadvantages of current polysaccharide-based vaccines, the most promising alternative for developing improved vaccines may be to use protein antigens with different roles in pneumococcus virulence. PspA and PhtD, highly immunogenic surface proteins expressed by almost all pneumococcal strains, are capable of eliciting protective immunity against lethal infections. Methods: In this study using immunoinformatics approaches, we constructed one fusion construct (called PAD) by fusing the immunodominant regions of PspA from families 1 & 2 (PA) to the immunodominant regions of PhtD (PD). The objective of this project was to test the immunogenicity of the fusion protein PAD and to compare its protective activity against S. pneumoniae infection with PA or PD alone and a combination of PA and PD. The prediction of physicochemical properties, antigenicity, allergenicity, toxicity, and 3D-structure of the constructs, as well as molecular docking with HLA receptor and immune simulation were performed using computational tools. Finally, mice were immunized and the serum levels of antibodies/cytokines and functionality of antibodies in vitro were evaluated after immunization. The mice survival rates and decrease of bacterial loads in the blood/spleen were examined following the challenge. Results: The computational analyses indicated the proposed constructs could be antigenic, non-allergenic, non-toxic, soluble and able to elicit robust immune responses. The results of actual animal experiments revealed the candidate vaccines could induce the mice to produce high levels of antibodies and cytokines. The complement-mediated bactericidal activity of antibodies was confirmed and the antibodies provided favorable survival in immunized mice after bacterial challenge. In general, the experimental results verified the immunoinformatics studies. Conclusion: For the first time this report presents novel peptide-based vaccine candidates consisting of immunodominant regions of PspA and PhtD antigens. The obtained findings confirmed that the fusion formulation could be relatively more efficient than the individual and combination formulations. The results propose that the fusion protein alone could be used as a serotype-independent pneumococcal vaccine or as an effective partner protein for a conjugate polysaccharide vaccine.

Development of a bivalent protein-based vaccine candidate against invasive pneumococcal diseases based on novel pneumococcal surface protein A in combination with pneumococcal histidine triad protein D.

Extensive efforts have been made toward improving effective strategies for pneumococcal vaccination, focusing on evaluating the potential of multivalent protein-based vaccines and overcoming the limitations of pneumococcal polysaccharide-based vaccines. In this study, we investigated the protective potential of mice co-immunization with the pneumococcal PhtD and novel rPspA proteins against pneumococcal sepsis infection. The formulations of each antigen alone or in combination were administered intraperitoneally with alum adjuvant into BALB/c mice three times at 14-day intervals. The production of antigen-specific IgG, IgG1 and IgG2a subclasses, and IL-4 and IFN-γ cytokines, were analyzed. Two in vitro complement- and opsonophagocytic-mediated killing activities of raised antibodies on day 42 were also assessed. Finally, the protection against an intraperitoneal challenge with 106 CFU/mouse of multi-drug resistance of Streptococcus pneumoniae ATCC49619 was investigated. Our findings showed a significant increase in the anti-PhtD and anti-rPspA sera IgG levels in the immunized group with the PhtD+rPspA formulation compared to each alone. Moreover, the results demonstrated a synergistic effect with a 6.7- and 1.3- fold increase in anti-PhtD and anti-rPspA IgG1, as well as a 5.59- and 1.08- fold increase in anti-PhtD and anti-rPspA IgG2a, respectively. Co-administration of rPspA+PhtD elicited a mixture of Th-2 and Th-1 immune responses, more towards Th-2. In addition, the highest complement-mediated killing activity was observed in the sera of the immunized group with PhtD+rPspA at 1/16 dilution, and the opsonophagocytic activity was increased from 74% to 86.3%. Finally, the survival rates showed that mice receiving the rPspA+PhtD formulation survived significantly longer (100%) than those receiving protein alone or PBS and exhibited the strongest clearance with a 2 log10 decrease in bacterial load in the blood 24h after challenge compared to the control group. In conclusion, the rPspA+PhtD formulation can be considered a promising bivalent serotype-independent vaccine candidate for protection against invasive pneumococcal infection in the future.

Evaluation of transmembrane domain deletions on hyaluronic acid polymerization of hyaluronan synthase isolated from Streptococcus equisimilis group G.

The membrane enzyme of hyaluronan synthase (HAS) is the key enzyme in hyaluronic acid (HA) biosynthesis by coupling UDP-sugars. Prior studies proposed the C-terminus region of HAS enzyme mediates the production rate and molecular weight of HA. The current study describes the isolation and characterizations of a transmembrane HAS enzyme isolated from Streptococcus equisimilis Group G (GGS-HAS) in vitro. The effect of transmembrane domains (TMDs) on HA productivity was determined and the shortest active variant was also identified by recombinant expression of full-length and five truncated forms of GGS-HAS in Escherichia coli. We found that the GGS-HAS enzyme is longer than that of S. equisimilis group C (GCS-HAS) which includes three more residues (LER) at the C-terminus region (positions 418-420) and also one-point mutation at position 120 (E120D). Amino acid sequence alignment demonstrated 98% and 71% identity of GGS-HAS with that of S. equisimilis Group C and S. pyogenes Group A, respectively. The in vitro productivity of the full-length enzyme was 35.57 µg/nmol, however, extended TMD deletions led to a reduction in the HA productivity. The HAS-123 variant showed the highest activity among the truncated forms, indicating the essential role of first, second, and third TMDs for the full activity. Despite a decline in activity, the intracellular variant can still mediate the binding and polymerization of HA without any need for TMDs. This significant finding suggests that the intracellular domain is the core for HA biosynthesis in the enzyme and other domains are probably involved in other attributes including the enzyme kinetics that affect the size distribution of the polymer. However, more investigations on the recombinant forms are still needed to confirm clearly the role of each transmembrane domain on these properties.

In-silico design and evaluation of an epitope-based serotype-independent promising vaccine candidate for highly cross-reactive regions of pneumococcal surface protein A.

BACKGROUND: The pathogenicity of pneumococcus with high morbidity, mortality, and multi-drug resistance patterns has been increasing. The limited coverage of the licensed polysaccharide-based vaccines and the replacement of the non-vaccine serotypes are the main reasons for producing a successful serotype-independent vaccine. Pneumococcal surface protein A (PspA) is an extremely important virulence factor and an interesting candidate for conserved protein-based pneumococcal vaccine classified into two prominent families containing five clades. PspA family-elicited immunity is clade-dependent, and the level of the PspA cross-reactivity is restricted to the same family. METHODS: To cover and overcome the clade-dependent immunity of the PspAs in this study, we designed and tested a PspA1-5c+p vaccine candidate composed of the highest immunodominant coverage of B- and T-cell epitope truncated domain of each clade focusing on two cross-reactive B and C regions of the PspAs. The antigenicity, toxicity, physicochemical properties, 3D structure prediction, stability and flexibility of the designed protein using molecular dynamic (MD) simulation, molecular docking of the construct withHLADRB1*(01:01) and human lactoferrin N-lop, and immune simulation were assessed using immunoinformatics tools. In the experimental section, after intraperitoneal immunization of the mice with Alum adjuvanted recombinant PspA1-5c+p, we evaluated the immune response, cross-reactivity, and functionality of the Anti-PspA1-5c+p antibody using ELISA, Opsonophagocytic killing activity, and serum bactericidal assay. RESULTS: For the first time, this work suggested a novel PspA-based vaccine candidate using immunoinformatics tools. The designed PspA1-5c+p protein is predicted to be highly antigenic, non-toxic, soluble, stable with low flexibility in MD simulation, and able to stimulate both humoral and cellular immune responses. The designed protein also could interact strongly with HLADRB1*(01:01) and human lactoferrin N-lop in the docking study. Our immunoinformatics predictions were validated using experimental data. Results showed that the anti-PspA1-5c+p IgG not only had a high titer with strong and same cross-reactivity coverage against all pneumococcal serotypes used but also had high and effective bioactivity for pneumococcal clearance using complement system and phagocytic cells. CONCLUSION: Our findings elucidated the potential application of the PspA1-5c+p vaccine candidate as a serotype-independent pneumococcal vaccine with a strong cross-reactivity feature. Further in-vitro and in-vivo investigations against other PspA clades should be performed to confirm the full protection of the PspA1-5c+p vaccine candidate.

Prevalence of Facial Asymmetry and Correction Methods for Rhinoplasty in Individuals with Deviated Nose: A Brief Review.

Background: Surgeons frequently perform rhinoplasty on individuals who have facial asymmetry. Patients' discontent following rhinoplasty has been linked to facial asymmetry. On the other hand, correction of a deviated nose is a tough procedure, and it is not the same as septal deviation correction. Surgeons, who often perform rhinoplasty for deviated nose in people with asymmetrical faces, focus primarily on correcting nasal defects and overlook such facial asymmetry. Aim: We aimed to summarize and review the prevalence of facial asymmetry in patients subjected for rhinoplasty for deviated nose correction. Methods: A systematic search was conducted covering PubMed, Scopus, ISI, and Google Scholar using related key words and MeSH (Medical Subject Headings) terms from 2000 until November 2021 for English published articles. Results: The majority of subjects had more facial asymmetry such as chin deviation, nasal deviation, and face breadth. Facial asymmetry is typically found in patients undergoing rhinoplasty for a deviated nose, and its presence frequently results in the failure to achieve a straight-looking nose. Conclusion: Patients considering rhinoplasty frequently have facial asymmetries, and careful attention should be devoted to these elements in both surgical planning and patient counseling. In order to create facial harmony and apparent symmetry after rhinoplasty, it is critical to center the nose on the midglabellar to mid-bow Cupid's line.

Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase.

BACKGROUND: Pseudomonas putida (P. putida) ATCC12633 can produce creatinase. It is a microbial enzyme which degrades creatinine in bacteria and provides source of carbon and nitrogen. Also, this enzyme is used in the enzymatic measurement of creatinine concentration for diagnosis of renal and muscles functions and diseases. Our purpose was recombinant production of creatinase for using in clinical measurement of serum or urine creatinine. METHODS: A 1209bp of open reading frame of creatinase was amplified by PCR from P. putida ATCC12633 genome and cloned into pET28a expression vector which was digested using NheI and XhoI restriction enzymes. Cloning was confirmed by colony PCR, double digestion analysis and sequencing. Recombinant pET28a vector was transformed to Escherichia coli (E. coli) BL21 (DE3). Creatinase expression was induced in E.coli BL21 (DE3) using IPTG and confirmed by SDS-PAGE and western blotting. Purification of creatinase was performed using Ni-NTA column. The specific activity of this enzyme was also investigated. RESULTS: The creatinase gene cloning was confirmed by DNA sequencing. Successful expression of creatinase was performed in E. coli (57.4% of total protein). SDS-PAGE and western blot analysis showed a 45 kDa creatinase protein. Purification of creatinase was done with high purity. The specific activity of recombinant enzyme is 26.54 unit/mg that is much higher than other creatinase used in the commercial kits (9 unit/mg). CONCLUSION: The P. putida ATCC12633 recombinant creatinase was expressed efficiently in E. coli BL21 and 57% of total protein was the recombinant creatinase. Also, expressed creatinase has high solubility and also the enzyme has good activity compared to enzymes used in commercial kits, so a new source of creatinase was produced for creatinine assay kit in this study.

A novel laparoscopic grasper with two parallel jaws capable of extracting the mechanical behaviour of soft tissues.

Data related to force-deformation behaviour of soft tissue plays an important role in medical/surgical applications such as realistically modelling mechanical behaviour of soft tissue as well as minimally invasive surgery (MIS) and medical diagnosis. While the mechanical behaviour of soft tissue is very complex due to its different constitutive components, some issues increase its complexity like behavioural changes between the live and dead tissues. Indeed, an adequate quantitative description of mechanical behaviour of soft tissues requires high quality in vivo experimental data to be obtained and analysed. This paper describes a novel laparoscopic grasper with two parallel jaws capable of obtaining compressive force-deformation data related to mechanical behaviour of soft tissues. This new laparoscopic grasper includes four sections as mechanical hardware, sensory part, electrical/electronical part and data storage part. By considering a unique design for mechanical hardware, data recording conditions will be close to unconfined-compression-test conditions; so obtained data can be properly used in extracting the mechanical behaviour of soft tissues. Also, the other distinguishing feature of this new system is its applicability during different laparoscopic surgeries and subsequently obtaining in vivo data. However, more preclinical examinations are needed to evaluate the practicality of the novel laparoscopic grasper with two parallel jaws.

Review on different experimental techniques developed for recording force-deformation behaviour of soft tissues; with a view to surgery simulation applications.

Different experimental techniques which have been developed to obtain data related to force-deformation behaviour of soft tissues play an important role in realistically simulating surgery processes as well as medical diagnoses and minimally invasive procedures. Indeed, an adequate quantitative description of soft-tissue-mechanical-behaviour requires high-quality experimental data to be obtained and analysed. In this review article we will first scan the motivations and basic technical issues on surgery simulation. Then, we will concentrate on different experimental techniques developed for recording force-deformation (stress-strain) behaviour of soft tissues with focussing on the in-vivo experimental setups. We will thoroughly review the available techniques by classifying them to four groups; elastography, indentation, aspiration and grasping techniques. The evolutions, advantages and limitations of each technique will be presented by a historical review. At the end, a discussion is given with the aim of summarising the proposed points and predicting the future of techniques utilised in extracting data related to force-deformation behaviour.

The relationship of serum vitamin D and Zinc in a nationally representative sample of Iranian children and adolescents: The CASPIAN-III study.

Background: Vitamin D (VitD) deficiency is a common worldwide problem. Some previous studies have shown that both Zinc (Zn) and VitD deficiency are prevalent in Iran. This study aimed to assess the relationship of serum Zn and vitamin D levels in a nationally representative sample of Iranian children and adolescents. Methods: This case-control study was conducted as a sub-study of a school-based surveillance program entitled "the CASPIAN-III Study". An equal number of individuals with and without hypovitaminosis D including 330 participants aged 10 to 18 years were selected. The correlation of serum 25 hydroxy vitamin D (25(OH) D), cardiometabolic factors and Zn concentrations was determined. Statistical analysis was done using one-way analysis of variance (ANOVA), Pearson's correlation, linear regression, and logistic regression. Results: The mean age was not significantly different in participants with and without hypovitaminosis D (14.74±2.52 vs. 14.74±2.66 years, respectively, p>0.05). The mean 25(OH) D level was 6.34±1.47ng/ml in the group with hypovitaminosis D and 39.27±6.42ng/ml in controls. The mean Zn level was significantly lower in the hypovitaminosis D group than in controls (1.15±0.26 vs. 1.43±0.32µg/ml, respectively, p<0.001). The Pearson's analysis showed a positive and significant correlation between Zn and 25(OH) D serum levels (p<0.0001). Odds ratios analysis for VitD level between various quartiles of serum zinc concentration for all participants showed that the odds of higher levels of VitD increased by higher levels of Zn. Conclusion: We found significant associations between low serum concentrations of zinc and 25(OH) D. Food fortification or mineral supplementation should be considered in future health programs.

Artery cross-clamping during laparoscopic vascular surgeries; a computational tactile sensing approach.

Artificial palpation is one of the most valuable achievements of artificial tactile sensing approach that can be used in various fields of medicine and more specifically in surgery. These techniques cause different surgical maneuvers to be done more precisely and noninvasively. In this study, considering the present problems and limitations of cross-clamping an artery during laparoscopic vascular surgeries, a new tactile sensory system will be introduced.Having imitated surgeon's palpation during open vascular surgeries and modeled it conceptually, the optimal amount of the total angular displacement of each robot joint in order to cross-clamping an artery without damaging to the artery surrounding tissues will be calculated. The elastic governing equation of contact occurred between the tactile sensor placed on the first link of the robot and the surrounding tissues around the artery were developed. A finite element model is coupled with genetic algorithm optimization method so that the normal stress and displacements in contact surface of the robot and artery's surrounding tissues would be minimized. Thus, reliability and accuracy of artificial tactile sensing method in artery cross-clamping will be demonstrated. Finally, the functional principles of the new tactile system capable of cross-clamping an artery during laparoscopic surgeries will be presented.

Design and fabrication of a novel tactile sensory system applicable in artificial palpation.

Force and position feedback are the two important parameters that are employed in different medical diagnoses and more specifically surgical operations. Furthermore, during different minimally invasive procedures, the ability of touch and force and position feedback are absent. In this regard, artificial palpation is a new technology that is employed to obtain tactile data in situations where physicians/surgeons cannot use their tactile sense. One of the most valuable achievements of artificial palpation are tactile sensory systems that have various applications in the detection of hard objects inside the soft tissue. Considering the present problems and limitations of kidney stone removal laparoscopy, the aim of this research is to design and fabricate a novel tactile sensory system capable of determining the exact location of stones during laparoscopy. This new tactile sensory system consists of four main parts: The sensory part, the mechanical part, the electrical part, and the display part. In this new system, due to the use of both displacement and force sensors, the usage limitations of previous tactile sensory systems are eliminated. The new tactile sensory system is well capable of finding the stone in the laboratory models through physical contact with the model's surface.

Application of artificial tactile sensing approach in kidney-stone-removal laparoscopy.

Artificial tactile sensing is a novel method for obtaining different characteristics of a hard object embedded in a soft tissue. In this regard, artificial palpation is one of the most valuable achievements of artificial tactile sensing that can be used in various fields of medicine and more specifically in surgery. In this study, considering the present problems and limitations in kidney-stone-removal laparoscopy, a new application will be presented for artificial tactile sensing approach. Having imitated surgeon's palpation during open surgery and modeled it conceptually, indications of stone existence that appear on the surface of kidney (due to exerting mechanical load) were determined. A number of different cases were created and solved by the software. Using stress distribution contours and stress graphs, it is illustrated that the created stress patterns on the surface of kidney not only show the existence of stone inside, but also its exact location. In fact, the reliability and accuracy of artificial tactile sensing method in detection of kidney stone during laparoscopy is demonstrated by means of finite element analysis. Also, in this paper, the functional principles of tactile system capable of determining the exact location of stone during laparoscopy will be presented.