In this paper we report the differentiating properties of platelet-rich plasma releasates (PRPr) on human chondrocytes within elastomeric polycaprolactone triol-citrate (PCLT-CA) porous scaffold. Human-derived chondrocyte cellular content of glycosaminoglycans (GAGs) and total collagen were determined after seeding into PCLT-CA scaffold enriched with PRPr cells. Immunostaining and real time PCR was applied to evaluate the expression levels of chondrogenic and extracellular gene markers. Seeding of chondrocytes into PCLT-CA scaffold enriched with PRPr showed significant increase in total collagen and GAGs production compared with chondrocytes grown within control scaffold without PRPr cells. The mRNA levels of collagen II and SOX9 increased significantly while the upregulation in Cartilage Oligomeric Matrix Protein (COMP) expression was statistically insignificant. We also report the reduction of the expression levels of collagen I and III in chondrocytes as a consequence of proximity to PRPr cells within the scaffold. Interestingly, the pre-loading of PRPr caused an increase of expression levels of following extracellular matrix (ECM) proteins: fibronectin, laminin and integrin β over the period of 3 days. Overall, our results introduce the PCLT-CA elastomeric scaffold as a new system for cartilage tissue engineering. The method of PRPr cells loading prior to chondrocyte culture could be considered as a potential environment for cartilage tissue engineering as the differentiation and ECM formation is enhanced significantly.
Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses transmitted to humans by their common vector, Aedes mosquitoes. DENV infection represents one of the most widely spread mosquito-borne diseases whereas ZIKV infection occasionally re-emerged in the past causing outbreaks. Although there have been considerable advances in understanding the pathophysiology of these viruses, no effective vaccines or antiviral drugs are currently available. In this study, we evaluated the antiviral activity of carnosine, an endogenous dipeptide (β-alanyl-l-histidine), against DENV serotype 2 (DENV2) and ZIKV infection in human liver cells (Huh7). Computational studies were performed to predict the potential interactions between carnosine and viral proteins. Biochemical and cell-based assays were performed to validate the computational results. Mode-of-inhibition, plaque reduction, and immunostaining assays were performed to determine the antiviral activity of carnosine. Exogenous carnosine showed minimal cytotoxicity in Huh7 cells and rescued the viability of infected cells with EC50 values of 52.3 and 59.5 μM for DENV2 and ZIKV infection, respectively. Based on the mode-of-inhibition assays, carnosine inhibited DENV2 mainly by inhibiting viral genome replication and interfering with virus entry. Carnosine antiviral activity was verified with immunostaining assay where carnosine treatment diminished viral fluorescence signal. In conclusion, carnosine exhibited significant inhibitory effects against DENV2 and ZIKV replication in human liver cells and could be utilized as a lead peptide for the development of effective and safe antiviral agents against DENV and ZIKV.
N-methyl-D-aspartate receptors (NMDAR) play a central role in epileptogensis and NMDAR antagonists have been shown to have antiepileptic effects in animals and humans. Despite significant progress in the development of antiepileptic therapies over the previous 3 decades, a need still exists for novel therapies. We screened an in-house library of small molecules targeting the NMDA receptor. A novel indolyl compound, 2-(1,1-Dimethyl-1,3-dihydro-benzo[e]indol-2-ylidene)-malonaldehyde, (DDBM) showed the best binding with the NMDA receptor and computational docking data showed that DDBM antagonised the binding sites of the NMDA receptor at lower docking energies compared to other molecules. Using a rat electroconvulsive shock (ECS) model of epilepsy we showed that DDBM decreased seizure duration and improved the histological outcomes. Our data show for the first time that indolyls like DDBM have robust anticonvulsive activity and have the potential to be developed as novel anticonvulsants.
Zika virus (ZIKV) represents a re-emerging threat to global health due to its association with congenital birth defects. ZIKV NS2B-NS3 protease is crucial for virus replication by cleaving viral polyprotein at various junctions to release viral proteins and cause cytotoxic effects in ZIKV-infected cells. This study characterized the inhibitory effects of doxycycline against ZIKV NS2B-NS3 protease and viral replication in human skin cells. The in silico data showed that doxycycline binds to the active site of ZIKV protease at a low docking energy (-7.8 Kcal/mol) via four hydrogen bonds with the protease residues TYR1130, SER1135, GLY1151, and ASP83. Doxycycline efficiently inhibited viral NS2B-NS3 protease at average human temperature (37 °C) and human temperature with a high fever during virus infection (40 °C). Interestingly, doxycycline showed a higher inhibitory effect at 40 °C (IC50 = 5.3 µM) compared to 37 °C (9.9 µM). The virus replication was considerably reduced by increasing the concentration of doxycycline. An approximately 50% reduction in virus replication was observed at 20 µM of doxycycline. Treatment with 20 µM of doxycycline reduced the cytopathic effects (CPE), and the 40 µM of doxycycline almost eliminated the CPE of human skin cells. This study showed that doxycycline binds to the ZIKV protease and inhibits its catalytic activity at a low micro-molecular concentration range. Treatment of human skin fibroblast with doxycycline eliminated ZIKV infection and protected the cells against the cytopathic effects of the infection.
Platelet rich plasma clot- releasate (PRCR) shows significant influence on tissue regeneration in clinical trials. Although, the mechanism of PRCR effect on fibroblast differentiation has been studied on 2D culture system, a detailed investigation is needed to establish the role of PRCR in cell seeded in 3D scaffolds. Therefore, a study was conducted to evaluate the influence of PRCR in fibroblasts (DFB) differentiation and extracellular matrix formation on both 3D and 2D culture systems. Cell viability was measured using MTT assay and DFB differentiation was evaluated by determining the expression levels of nucleostamin and alpha smooth muscle actin (α-SMA), using indirect immunostaining and Western blotting. The expression levels of extracellular matrix genes (collagen-I, collagen-III, fibronectin and laminin) and focal adhesion formation gene (integrin beta-1) were measured using Real-time PCR. The PRCR at 10% showed significant effect on cells viability compared with 5% and 20% in both culture environments. The decrease in the expression levels of nucleostamin and the increase in α-SMA signify the DFB differentiation to myofibroblast-like cells that was prominently greater in 3D compared to 2D culture. In 3D culture systems, the total collage production, expression levels of the extracellular matrix gene and the focal adhesion gene were increased significantly compared to 2D culture. In conclusion, 3D culture environments enhances the proliferative and differentiation effects of PRCR on DFB, thereby potentially increases the efficacy of DFB for future tissue engineering clinical application.
Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.
Global resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern. Therefore, there is an urgent need for the development of successful drugs that are both economical and offer a long-lasting protection. The viral NS2B-NS3 serine protease (NS2B-NS3pro) is a promising target for the development of drug-like inhibitors, which are not available at the moment. In this study, we report retrocyclin-1 (RC-1) production in E. coli as a recombinant peptide to test against dengue NS2B-NS3pro.
Dengue diseases have an economic as well as social burden worldwide. In this study, the antiviral activity of protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) peptide towards dengue NS2B-NS3pro and viral replication in Rhesus monkey kidney (MK2) cells was investigated. The peptide PG-1 was synthesized by solid-phase peptide synthesis, and disulphide bonds formation followed by peptide purification was confirmed by LC-MS and RPHPLC. Dengue NS2B-NS3pro was produced as a single-chain recombinant protein in E. coli. The NS2B-NS3pro assay was carried out by measuring the florescence emission of catalyzed substrate. Real-time PCR was used to evaluate the inhibition potential of PG-1 towards dengue serotype-2 (DENV-2) replication in MK2 cells. The results showed that PG-1 inhibited dengue NS2B-NS3pro at IC(50) of 11.7 μM. The graded concentrations of PG-1 at nontoxic range were able to reduce viral replication significantly (P < 0.001) at 24, 48, and 72 hrs after viral infection. However, the percentage of inhibition was significantly (P < 0.01) higher at 24 hrs compared to 48 and 72 hrs. These data show promising therapeutic potential of PG-1 against dengue infection, hence it warrants further analysis and improvement of the peptide features as a prospective starting point for consideration in designing attractive dengue virus inhibitors.
Chondrogenic differentiated mesenchymal stem cells (CMSCs) have been shown to produce superior chondrogenic expression markers in vitro. However, the use of these cells in vivo has not been fully explored. In this study, in vivo assessment of cartilage repair potential between allogenic-derived chondrogenic pre-differentiated mesenchymal stem cells and undifferentiated MSCs (MSCs) were compared. Bilateral full thickness cartilage defects were created on the medial femoral condyles of 12 rabbits (n = 12). Rabbits were divided into two groups. In one group, the defects in the right knees were repaired using alginate encapsulated MSCs while in the second group, CMSCs were used. The animals were sacrificed and the repaired and control knees were assessed at 3 and 6 months after implantation. Quantitative analysis was performed by measuring the Glycosaminoglycans (GAGs)/total protein content. The mean Brittberg score was higher in the transplanted knees as compared to the untreated knee at 6 months (p 0.05). This study demonstrates that the use of either MSC or CMSC produced superior healing when compared to cartilage defects that were untreated. However, both cells produced comparable treatment outcomes.
Dengue is the current leading cause of death among children in several Latin American and Asian countries. Due to poverty in areas where the disease is prevalent and the high cost of conventional diagnostic systems, low cost devices are needed to reduce the burden caused by dengue infection. Centrifugal microfluidic platforms are an alternative solution to reduce costs and increase the availability of a rapid diagnostic system. The rate of chemical reactions in such devices often depends on the efficiency of the mixing techniques employed in their microfluidic networks. This paper introduces a micromixer that operates by the expansion and contraction of a microballoon to produce a consistent periodical 3D reciprocating flow. We established that microballoons reduced mixing time of 12 μl liquids from 170 min, for diffusional mixing, to less than 23 s. We have also tested the effect of the microballoon mixers on the detection of the dengue virus. The results indicate that employing a microballoon mixer enhances the detection sensitivity of the dengue virus by nearly one order of magnitude compared to the conventional ELISA method.
Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have led to significant impact on health care in the developing world. Here, we evaluated the antiviral effects of tetracycline (TETRA) derivatives and other common antiviral agents against CHIKV. Our results showed that within the TETRA derivatives group, Doxycycline (DOXY) exhibited the highest inhibitory effect against CHIKV replication in Vero cells. On the other hand, in the antiviral group Ribavirin (RIBA) showed higher inhibitory effects against CHIKV replication compared to Aciclovir (ACIC). Interestingly, RIBA inhibitory effects were also higher than all but DOXY within the TETRA derivatives group. Docking studies of DOXY to viral cysteine protease and E2 envelope protein showed non-competitive interaction with docking energy of -6.6±0.1 and -6.4±0.1 kcal/mol respectively. The 50% effective concentration (EC50) of DOXY and RIBA was determined to be 10.95±2.12 μM and 15.51±1.62 μM respectively, while DOXY+RIBA (1:1 combination) showed an EC50 of 4.52±1.42 μM. When compared, DOXY showed higher inhibition of viral infectivity and entry than RIBA. In contrast however, RIBA showed higher inhibition against viral replication in target cells compared to DOXY. Assays using mice as animal models revealed that DOXY+RIBA effectively inhibited CHIKV replication and attenuated its infectivity in vivo. Further experimental and clinical studies are warranted to investigate their potential application for clinical intervention of CHIKV disease.
Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.
Centrifugal microfluidic systems utilize a conventional spindle motor to automate parallel biochemical assays on a single microfluidic disk. The integration of complex, sequential microfluidic procedures on these platforms relies on robust valving techniques that allow for the precise control and manipulation of fluid flow. The ability of valves to consistently return to their former conditions after each actuation plays a significant role in the real-time manipulation of fluidic operations. In this paper, we introduce an active valving technique that operates based on the deflection of a latex film with the potential for real-time flow manipulation in a wide range of operational spinning speeds. The reversible thermo-pneumatic valve (RTPV) seals or reopens an inlet when a trapped air volume is heated or cooled, respectively. The RTPV is a gas-impermeable valve composed of an air chamber enclosed by a latex membrane and a specially designed liquid transition chamber that enables the efficient usage of the applied thermal energy. Inputting thermo-pneumatic (TP) energy into the air chamber deflects the membrane into the liquid transition chamber against an inlet, sealing it and thus preventing fluid flow. From this point, a centrifugal pressure higher than the induced TP pressure in the air chamber reopens the fluid pathway. The behaviour of this newly introduced reversible valving system on a microfluidic disk is studied experimentally and theoretically over a range of rotational frequencies from 700 RPM to 2500 RPM. Furthermore, adding a physical component (e.g., a hemispherical rubber element) to induce initial flow resistance shifts the operational range of rotational frequencies of the RTPV to more than 6000 RPM. An analytical solution for the cooling of a heated RTPV on a spinning disk is also presented, which highlights the need for the future development of time-programmable RTPVs. Moreover, the reversibility and gas impermeability of the RTPV in the microfluidic networks are validated on a microfluidic disk designed for performing liquid circulation. Finally, an array of RTPVs is integrated into a microfluidic cartridge to enable sequential aliquoting for the conversion of dengue virus RNA to cDNA and the preparation of PCR reaction mixtures.
Road traffic accidents constitute the primary cause of fatalities associated with injuries and engender substantial economic ramifications for affected individuals, their families, and entire nations. The Sultanate of Oman, like other countries, suffers from traffic accident injuries and traffic congestion. The accident rate for the period 2021 was recorded as one accident every six hours. Despite a 70% increase in total number of vehicles and an 81% rise in licensed drivers between 2012 and 2019, data on traffic accidents demonstrate an improving trend with a notable 55% decline in crash fatalities. However, it is important to recognize that road traffic accidents in Oman encompass not only social issues but also pose a significant economic burden, resulting in substantial financial costs for the nation. Notwithstanding, it was discovered that more than 50% of fatal crashes in Oman were primarily caused by excessive speeding. The main goal of this research is to analysis the causes and trends of traffic accidents at the national level in the Sultanate of Oman. Data analysis reveals speed as the primary cause of traffic accidents in Oman, with Muscat and Dhofar registering the highest accident rates. In addition, the distribution of deaths and injuries resulting from accidents varies according to Gender and nationality. According to the road accident scenario analysis in the state, more traffic accidents occurred in males than females. Traffic accidents have witnessed a notable decline over the past decade, attributable to the diligent efforts and interventions implemented by the Royal Oman Police.
Three main types of PVC solvent polymeric membrane ion-selective electrodes for chloroquine are described. They are based on three ion-pairing agents namely dipicrylamine (DPA), tetraphenylborate (TPB) or tetrakis(4-chlorophenyl)borate (TCPB) with either dioctylphenyl phosphonate (DOPP) or trioctyl phosphate (TOP) solvent mediator. All electrodes exhibit Nernstian responses, fast dynamic response times and a wide useful pH range. The best all-round electrode is based on TPB and TOP plasticizing solvent mediators with a limit of detection of 7.1 x 10(-6)M and was utilized for the assay of chloroquine in tablets. Direct potentiometric determinations with either the analyte addition method or the normal calibration method gave results comparable to the official method.
Coronary sinus perforation is a relatively uncommon but much feared complication that may occur during the placement of left ventricular pacing lead. Coronary sinus perforation, especially in the presence of an obstructive flap, usually indicates the need to abandon the implantation attempt, as there are difficulties in crossing the obstructive flap as well as uncertainty of whether the lead is in the true lumen or into the pericardial space. We describe our experience in successfully placing the left ventricular lead safely despite the problems arising from these circumstances.
Two major flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), cause severe health and economic burdens worldwide. Recently, genome-wide screenings have uncovered the importance of regulators of the Hrd1 ubiquitin ligase-mediated endoplasmic reticulum (ER)-associated degradation (ERAD) pathway for flavivirus replication in host cells. Here we report the identification of the compound Bardoxolone methyl (CDDO-me) as a potent inhibitor of the Hrd1 ubiquitin ligase-mediated ERAD, which possesses a broad-spectrum activity against both DENV and ZIKV. Cellular thermal shift assay (CETSA) suggested that CDDO-me binds to grp94, a key component of the Hrd1 pathway, at a low nanomolar concentration, whereas interaction was not detected with its paralog Hsp90. CDDO-me and the grp94 inhibitor PU-WS13 substantially suppressed DENV2 replication and the cytopathic effects caused by DENV and ZIKV infection. The antiviral activities of both compounds were demonstrated for all four DENV serotypes and four ZIKV strains in multiple human cell lines. This study defines grp94 as a crucial host factor for flavivirus replication and identified CDDO-me as a potent small molecule inhibitor of flavivirus infection. Inhibition of grp94 may contribute to the antiviral activity of CDDO-me. Further investigation of grp94 inhibitors may lead to a new class of broad-spectrum anti-flaviviral medications.
Pasteurella multocida is the main cause of haemorrhagic septicaemia (HS) outbreak in livestock, such as cattle and buffaloes. Conventional vaccines such as alum-precipitated or oil-adjuvant broth bacterins were injected subcutaneously to provide protection against HS. However, the immunity developed is only for short term and needed to be administered frequently. In our previous study, a short gene fragment from Pasteurella multocida serotype B was obtained via shotgun cloning technique and later was cloned into bacterial expression system. pQE32-ABA392 was found to possess immunogenic activity towards HS when tested in vivo in rat model. In this study, the targeted gene fragment of ABA392 was sub-cloned into a DNA expression vector pVAX1 and named as pVAX1-ABA392. The new recombinant vaccine was stable and expressed on mammalian cell lines. Serum sample collected from a group of vaccinated rats for ELISA test shows that the antibody in immunized rats was present at high titer and can be tested as a vaccine candidate with challenge in further studies. This successful recombinant vaccine is immunogenic and potentially could be used as vaccine in future against HS.
Electrospun polyhydroxybutyrate (PHB) fibers were dip-coated by polymethyl methacrylate-co-methacrylic acid, poly(MMA-co-MAA), which was synthesized in different molar ratios of the monomers via free-radical polymerization. Fabricated platfrom was employed for immobilization of the dengue antibody and subsequent detection of dengue enveloped virus in enzyme-linked immunosorbent assay (ELISA). There is a major advantage for combination of electrospun fibers and copolymers. Fiber structre of electrospun PHB provides large specific surface area available for biomolecular interaction. In addition, polymer coated parts of the platform inherited the premanent presence of surface carboxyl (-COOH) groups from MAA segments of the copolymer which can be effectively used for covalent and physical protein immobilization. By tuning the concentration of MAA monomers in polymerization reaction the concentration of surface -COOH groups can be carefully controlled. Therefore two different techniques have been used for immobilization of the dengue antibody aimed for dengue detection: physical attachment of dengue antibodies to the surface and covalent immobilization of antibodies through carbodiimide chemistry. In that perspective, several different characterization techniques were employed to investigate the new polymeric fiber platform such as scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle (WCA) measurement and UV-vis titration. Regardless of the immobilization techniques, substantially higher signal intensity was recorded from developed platform in comparison to the conventional ELISA assay.
In this paper, we propose an easy-to-implement passive liquid valve (PLV) for the microfluidic compact-disc (CD). This valve can be implemented by introducing venting chambers to control the air flow of the source and destination chambers. The PLV mechanism is based on equalizing the main forces acting on the microfluidic CD (i.e., the centrifugal and capillary forces) to control the burst frequency of the source chamber liquid. For a better understanding of the physics behind the proposed PLV, an analytical model is described. Moreover, three parameters that control the effectiveness of the proposed valve, i.e., the liquid height, liquid density, and venting chamber position with respect to the CD center, are tested experimentally. To demonstrate the ability of the proposed PLV valve, microfluidic liquid switching and liquid metering are performed. In addition, a Bradford assay is performed to measure the protein concentration and evaluated in comparison to the benchtop procedure. The result shows that the proposed valve can be implemented in any microfluidic process that requires simplicity and accuracy. Moreover, the developed valve increases the flexibility of the centrifugal CD platform for passive control of the liquid flow without the need for an external force or trigger.