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Background: Five-membered heterocycle with one neighbouring nitrogen atoms is present in the indole nucleus. Heterocyclic substances feature two adjacent benzene and pyrrole ring atoms. Indole are heterocyclic compounds with one oxygen ring that have a benzoannelated - pyrrole ring. Indole derivatives are highly valuable chemical synthesis and medicinal intermediates. Recent years have seen a surge in interest in the effective synthesis of indole blocks and associated functionalization's, particularly the alkylation of indoles. The two following factors will be the main emphasis of this review: i) Recent advancements in the alkylation of indoles, namely those involving the N1-, C2- and C3-positions and the synthesis of indoles, which was facilitated by transition metals. Materials and Methods: Benzoic acid and ethanoic acid, propionic acid, hydroxylamine, 2,5 Dichloro aniline are used for the synthesis of indole derivatives were used for the synthesis of indole. Results: Compared to indomethacin, the anti-inflammatory results for the substances or their derivatives like N2-(biphenyl-2-yl)-4-chloro-N1-(5-chloro-1H-indol-1-yl)-4-am inobenzene-1,2-diamine NA5 (Scheme 1B); N2-(biphenyl-2-yl)-4-chloro-N1-(5-chloro-1H-ind ol-1-yl)-3-aminobenzene-1,2-diamine NA6 (Scheme 1B) and N2-(biphenyl-2-yl)-4-chloro-N1-(5-chloro-1H-indol-1-yl)-2-aminobenzene-1,2-diamine NA7 (Scheme 1B). Conclusion: Examined were the anti-inflammatory properties of the substances in the title and their derivatives. The compounds containing indole derivatives with an electron-withdrawing group have a higher activity than those containing an electron-donating group, according to research on the link between structure and activity. Summarising the data from the previously reviewed literature, we can say that indole has a broad spectrum of biological activity. Numerous opportunities exist to investigate indole for new therapeutic uses. Researchers worldwide will benefit from this study's coverage of the chemistry of indole derivatives in the design and synthesis of new pharmaceuticals that might be utilised to treat a range of diseases. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic highlighted public awareness of airborne disease transmission in indoor settings and emphasized the need for reliable air disinfection technologies. This increased awareness will carry in the post-pandemic era along with the ever-emerging SARS-CoV variants, necessitating effective and well-defined protocols, methods, and devices for air disinfection. Ultraviolet (UV)-based air disinfection demonstrated promising results in inactivating viral bioaerosols. However, the reported data diversity on the required UVC doses has hindered determining the best UVC practices and led to confusion among the public and regulators. This article reviews available information on critical parameters influencing the efficacy of a UVC air disinfection system and, consequently, the required dose including the systems components as well as operational and environmental factors. There is a consensus in the literature that the interrelation of humidity and air temperature has a significant impact on the UVC susceptibility, which translate to changing the UVC efficacy of commercialized devices in indoor settings under varying conditions. Sampling and aerosolization techniques reported to have major influence on the result interpretation and it is recommended to use several sampling methods simultaneously to generate comparable and conclusive data. We also considered the safety concerns and the potential safe alternative of UVC, far-UVC. Finally, the gaps in each critical parameter and the future research needs of the field are represented. This paper is the first step to consolidating literature towards developing a standard validation protocol for UVC air disinfection devices which is determined as the one of the research needs.

In this study low-input RNA-sequencing was used to annotate the molecular identity of endothelial cells isolated and immunopurified with CD144 microbeads. Using this technique, comparative gene expression profiling from healthy subjects and patients with type 2 diabetes mellitus identified both known and novel pathways linked with EC dysfunction. Modeling of diabetes by treating cultured ECs with high glucose identified shared changes in gene expression in diabetic cells. Overall, the data demonstrate how purified ECs from patients can be used to generate new hypotheses about mechanisms of human vascular disease.

α-Synuclein is a protein linked to various synuclein-associated diseases ('synucleinopathies'), including Parkinson's disease, dementia with Lewy Bodies and multiple system atrophy, and is highly expressed in the central nervous system and in erythrocytes. Moreover, α-synuclein-containing erythrocyte-derived extracellular vesicles may be involved in the pathogenesis of synucleinopathies and their progression across the blood–brain barrier. Several post-translational modifications of α-synuclein have been reported in brain inclusions, including S129 phosphorylation, but fewer have been found in erythrocytes. In this study, we analysed the post-translational modifications of erythrocyte α-synuclein using liquid chromatography–mass spectrometry. We found that all lysine residues in the α-synuclein protein could be modified by acetylation, glycation, ubiquitination or SUMOylation but that phosphorylation, nitration and acylation were uncommon minor post-translational modifications in erythrocytes. Since the post-translational modification of lysine residues has been implicated in both membrane association and protein clearance, our findings provide new insight into how synucleinopathies may progress and suggest possible therapeutic strategies designed to target α-synuclein. [ABSTRACT FROM AUTHOR]

Aims: To develop an early implant instability murine model and explore the use of intermittent parathyroid hormone (iPTH) treatment for initially unstable implants. Methods: 3D-printed titanium implants were inserted into an oversized drill-hole in the tibiae of C57Bl/6 mice (n = 54). After implantation, the mice were randomly divided into three treatment groups (phosphate buffered saline (PBS)-control, iPTH, and delayed iPTH). Radiological analysis, micro-CT (µCT), and biomechanical pull-out testing were performed to assess implant loosening, bone formation, and osseointegration. Peri-implant tissue formation and cellular composition were evaluated by histology. Results: iPTH reduced radiological signs of loosening and led to an increase in peri-implant bone formation over the course of four weeks (timepoints: one week, two weeks, and four weeks). Observational histological analysis shows that iPTH prohibits the progression of fibrosis. Delaying iPTH treatment until after onset of peri-implant fibrosis still resulted in enhanced osseointegration and implant stability. Despite initial instability, iPTH increased the mean pull-out strength of the implant from 8.41 N (SD 8.15) in the PBS-control group to 21.49 N (SD 10.45) and 23.68 N (SD 8.99) in the immediate and delayed iPTH groups, respectively. Immediate and delayed iPTH increased mean peri-implant bone volume fraction (BV/TV) to 0.46 (SD 0.07) and 0.34 (SD 0.10), respectively, compared to PBS-control mean BV/TV of 0.23 (SD 0.03) (PBS-control vs immediate iPTH, p < 0.001; PBS-control vs delayed iPTH, p = 0.048; immediate iPTH vs delayed iPTH, p = 0.111). Conclusion: iPTH treatment mediated successful osseointegration and increased bone mechanical strength, despite initial implant instability. Clinically, this suggests that initially unstable implants may be osseointegrated with iPTH treatment. Cite this article: Bone Joint Res 2022;11(5):260–269.

Mast cell (MC) degranulation has been implicated in the side effect profile of a variety of clinically useful agents. Thus, after intrathecal delivery, formation of space-occupying, meningeally-derived masses may be related to local MC degranulation. We systematically characterized degranulating effects of opioid and nonopioid analgesics on cutaneous flares in the dog and in primary human MC (hMC) cultures.MethodsDogs were anesthetized with IV propofol and received intradermal (ID) injections (50μL). Flare diameters were measured at 30min. Drugs showing flare responses were tested after intramuscular (IM) cromolyn (10mg/kg), a MC stabilizer. Human primary MCs (human cord blood CD34+/CD45+ cells) were employed and β-hexosaminidase in cell-free supernatants were measured to assess degranulation.ResultsA significant skin flare for several classes of agents was observed including opioids, ziconotide, ketamine, ST-91, neostigmine, adenosine, bupivacaine, lidocaine, MK-801 and 48/80. Tizanidine, fentanyl, alfentanil, gabapentin and baclofen produced no flare. Flare produced by all ID agents, except adenosine, bupivacaine and lidocaine, was reduced by cromolyn. Naloxone had no effect upon opiate or 48/80 evoked flares. In hMC studies, 48/80 resulted in a concentration-dependent release of β-hexosaminidase. The rank order of drug-induced hMC β-hexosaminidase release was similar to that for flares.ConclusionsA variety of therapeutically useful drugs degranulate MCs. This action may account for side effects such as the intrathecal granuloma resulting from spinally-delivered opioids. This degranulating effect may be useful in predicting potential intrathecal toxicity in the development of novel agents.

Background: Improving the early identification of HIV-1-infected newborns with birth testing is critical to comprehensive early infant diagnosis and care for newborns living with HIV-1. Automated RNA quantification systems are valuable diagnostic tools, but the volume of plasma that viral load platforms require makes their widespread use for young children difficult. Method: Seventy-nine plasma samples with different viral load ranges were evaluated in parallel with the use of 1x PBS, pH 7.4, to supplement the required volume at dilutions factors from 1:2 to 1:50. Viral load quantification assays were evaluated using ABBOTT Molecular platforms, USA. Results: Using 1x PBS, at 1:10 dilution (70 µL plasma in 630 µL 1x PBS), a sensitivity of 100% and 100% specificity were obtained for detecting a viremia above 400 copies/mL (Kappa of 0.96, p

BACKGROUND: The methods to extract human bone marrow mesenchymal stem cells, including whole bone marrow adherent culture, density gradient centrifugation, flow cytometry separation and immunomagnetic bead separation. Flow cytometry separation and magnetic bead separation are restricted on equipment and reagents, so they are limited to applied. The whole bone marrow adherent culture method and density gradient centrifugation method are commonly used; however, there are still some shortcomings, such as slow cell growth rate and long centrifugation time. Thus, the efficiency of the experiment will be further improved if a more convenient and short time extraction method of bone marrow mesenchymal stem cells is found. OBJECTIVE: To introduce a new method of extracting human bone marrow mesenchymal stem cells and compare the extracted cells with those extracted by traditional methods to prove the feasibility and practicability of the new method. METHODS: Phosphate buffered saline was mixed with human bone marrow tissue at the same ratio, and the mixture was stratified by centrifugation. Human bone marrow mesenchymal stem cells were extracted from the white membrane layer followed by comparison with the cells extracted using whole bone marrow adherent method. The extracted cells were identified as bone marrow mesenchymal stem cells by flow cytometry and multidirectional differentiation potential. The number of primary cells, cell morphology, size, cytoskeleton, expression of surface markers, multidirectional differentiation potential, colony formation, proliferation, migration, and osteogenic differentiation were analyzed by cell counting, crystal violet staining, cytoskeleton staining, scratch test, Transwell migration test, and RT-PCR. RESULTS AND CONCLUSION: (1) After centrifugation, human bone marrow samples were stratified using phosphate buffered saline to extract human bone marrow mesenchymal stem cells. The number of primary cells extracted by phosphate buffered saline method was more than that of whole bone marrow adherent method. The floating adipose tissue was less in the culture system, and the primary cells grew faster. (2) The cells extracted by phosphate buffered saline and whole bone marrow adhesion were fusiform and expressed CD90, CD105, and CD73, but did not express CD45, CD34, CD11b, CD19, and HLA-DR. They had multidirectional differentiation potential and met the standard of mesenchymal stem cells. (3) There was no significant difference in morphology, size and cytoskeleton of the cells extracted using the two methods. Both groups of cells could form colonies and had migration ability. After osteogenic differentiation, both groups of cells expressed osteoblast markers. (4) The results show that it is feasible and practical to extract human bone marrow mesenchymal stem cells with phosphate buffered saline under certain conditions. [ABSTRACT FROM AUTHOR]

Introduction: Furcation perforation is one of the complications that occur during endodontic treatment due to anatomical differences or iatrogenic causes. Repairing the perforation with a biocompatible material eliminates the connection with the gingival sulcus and positively affects the prognosis. The sealing ability of the repair materials is very important and is affected by the conditions of the environment in which they are applied. Aim: To compare the sealing efficiency of Mineral Trioxide Aggregate (MTA) (Angelus, Londrina, PR, Brazil) and Biodentine (Septodont, Saint Maur des Fosses, France) materials used in the repair of furcation perforations in acidic and neutral environments. Materials and Methods: This in-vitro experimental study was carried out in the Department of Endodontics at Dicle University Faculty of Dentistry, Diyarbakır, Türkiye and Dicle University Science and Technology Application and Research Center from 10th June 2020 to 22nd July 2020. A total of 96 mandibular molars were used in the study. The specimens were randomly divided into two equal groups (n=48). When the perforations were repaired with MTA and Biodentine materials, each group was divided into four subgroups (n=12). These subgroups were kept in Phosphate Buffered Saline (PBS) and Acetic Acid (AA) solutions for different periods of time from 4 or 34 days, and all groups were immersed in methylene blue solution. The dye penetration level of the divided sections was examined under a stereomicroscope (Leica, Wetzlar, Germany). Microleakage data of all groups were statistically analysed using Kolmogorov Smirnov, Shapiro Wilk’s and Mann-Whitney U Test. Statistical significance was set at 0.05, it was stated that there was a significant difference when p-value 0.05). The long-term microleakage level in PBS environment of biodentine material was significantly lower than that of the AA environment (p-value=0.008). Conclusion: Lower microleakage levels were observed in the neutral environment than the acid environment with both MTA and biodentine during both holding periods

Introduction: Furcation perforation is one of the complications that occur during endodontic treatment due to anatomical differences or iatrogenic causes. Repairing the perforation with a bio-compatible material eliminates the connection with the gingival sulcus and positively affects the prognosis. The sealing ability of the repair materials is very important and is affected by the conditions of the environment in which they are applied. Aim: To compare the sealing efficiency of Mineral Trioxide Aggregate (MTA) (Angelus, Londrina, PR, Brazil) and Biodentine (Septodont, Saint Maur des Fosses, France) materials used in the repair of furcation perforations in acidic and neutral environments. Materials and Methods: This in-vitro experimental study was carried out in the Department of Endodontics at Dicle University Faculty of Dentistry, Diyarbakır, Türkiye and Dicle University Science and Technology Application and Research Center from 10th June 2020 to 22nd July 2020. A total of 96 mandibular molars were used in the study. The specimens were randomly divided into two equal groups (n=48). When the perforations were repaired with MTA and Biodentine materials, each group was divided into four subgroups (n=12). These subgroups were kept in Phosphate-Buffered Saline (PBS) and Acetic Acid (AA) solutions for different periods of time from four or 34 days, and all groups were immersed in methylene blue solution. The dye penetration level of the divided sections was examined under a stereomicroscope (Leica, Wetzlar, Germany). Microleakage data of all groups were statistically analysed using Kolmogorov Smirnov, Shapiro Wilk's and Mann-Whitney U Test. Statistical significance was set at 0.05, it was stated that there was a significant difference when p-value <0.05. Results: The short-term (4 days) microleakage level of biodentine material in the AA environment was significantly lower than that of MTA material (p-value=0.005). There was no statistically significant difference between the materials in terms of long-term (34 days) microleakage levels in the AA environment (p-value>0.05). The long-term microleakage level in PBS environment of biodentine material was significantly lower than that of the AA environment (p-value=0.008). Conclusion: Lower microleakage levels were observed in the neutral environment than the acid environment with both MTA and biodentine during both holding periods. [ABSTRACT FROM AUTHOR]

Clinical studies have indicated an association between acute hyperglycemia and poor outcomes in patients with traumatic brain injury (TBI), although optimal blood glucose levels needed to maximize outcomes for these patients' remain under investigation. Previous results from experimental animal models suggest that post-TBI hyperglycemia may be harmful, neutral, or beneficial. The current studies determined the effects of single or multiple episodes of acute hyperglycemia on cerebral glucose metabolism and neuronal injury in a rodent model of unilateral controlled cortical impact (CCI) injury. In Experiment 1, a single episode of hyperglycemia (50% glucose at 2 g/kg, i.p.) initiated immediately after CCI was found to significantly attenuate a TBI-induced depression of glucose metabolism in cerebral cortex (4 of 6 regions) and subcortical regions (2 of 7) as well as to significantly reduce the number of dead/dying neurons in cortex and hippocampus at 24 h post-CCI. Experiment 2 examined effects of more prolonged and intermittent hyperglycemia induced by glucose administrations (2 g/kg, i.p.) at 0, 1, 3 and 6h post-CCI. The latter study also found significantly improved cerebral metabolism (in 3 of 6 cortical and 3 of 7 subcortical regions) and significant neuroprotection in cortex and hippocampus 1 day after CCI and glucose administration. These results indicate that acute episodes of post-TBI hyperglycemia can be beneficial and are consistent with other recent studies showing benefits of providing exogenous energy substrates during periods of increased cerebral metabolic demand.

A significant proportion (estimates range from 16 to 74%) of patients with spinal cord injury (SCI) have concomitant traumatic brain injury (TBI), and the combination often produces difficulties in planning and implementing rehabilitation strategies and drug therapies. For example, many of the drugs used to treat SCI may interfere with cognitive rehabilitation, and conversely drugs that are used to control seizures in TBI patients may undermine locomotor recovery after SCI. The current paper presents an experimental animal model for combined SCI and TBI to help drive mechanistic studies of dual diagnosis. Rats received a unilateral SCI (75 kdyn) at C5 vertebral level, a unilateral TBI (2.0 mm depth, 4.0 m/s velocity impact on the forelimb sensori-motor cortex), or both SCI+TBI. TBI was placed either contralateral or ipsilateral to the SCI. Behavioral recovery was examined using paw placement in a cylinder, grooming, open field locomotion, and the IBB cereal eating test. Over 6weeks, in the paw placement test, SCI+contralateral TBI produced a profound deficit that failed to recover, but SCI+ipsilateral TBI increased the relative use of the paw on the SCI side. In the grooming test, SCI+contralateral TBI produced worse recovery than either lesion alone even though contralateral TBI alone produced no observable deficit. In the IBB forelimb test, SCI+contralateral TBI revealed a severe deficit that recovered in 3 weeks. For open field locomotion, SCI alone or in combination with TBI resulted in an initial deficit that recovered in 2 weeks. Thus, TBI and SCI affected forelimb function differently depending upon the test, reflecting different neural substrates underlying, for example, exploratory paw placement and stereotyped grooming. Concurrent SCI and TBI had significantly different effects on outcomes and recovery, depending upon laterality of the two lesions. Recovery of function after cervical SCI was retarded by the addition of a moderate TBI in the contralateral hemisphere in all tests, but forepaw placements were relatively increased by an ipsilateral TBI relative to SCI alone, perhaps due to the dual competing injuries influencing the use of both forelimbs. These findings emphasize the complexity of recovery from combined CNS injuries, and the possible role of plasticity and laterality in rehabilitation, and provide a start towards a useful preclinical model for evaluating effective therapies for combine SCI and TBI.

Abstract Background Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a contagious infectious disease that affects domestic and wild ruminants causing chronic inflammation of the intestine. MAP has proven to be very resistant to both physical and chemical processes, making it difficult to control this pathogen. Based on the recognized antimicrobial properties of copper, the objective of this study was to evaluate the effectiveness of copper ions to reduce MAP numbers and/or MAP viability in a fluid matrix. Besides, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli were used as controls of the effectiveness of copper ions. MAP-spiked PBS was subjected to copper ions treatment at 24 V for 5 min and the PBS suspensions were sampled before and after treatment. MAP viability and quantification were determined using three complementary techniques: a phage amplification assay, MGIT culture and qPCR. Results Moderate numbers (103 CFU ml−1) of the two control bacteria were completely eliminated by treatment with copper ions. For MAP, copper ions treatment reduced both the viability and numbers of this pathogen. Phage assay information quickly showed that copper ions (24 V for 5 min) resulted in a significant reduction in viable MAP. MGIT culture results over time showed statistically significant differences in time-to-detection (TTD) values between PRE and POST treatment. MAP genome equivalent estimates for PBS suspensions indicated that MAP numbers were lower in samples POST-treatment with copper ions than PRE-treatment. Conclusions The use of copper ions resulted in a significant reduction of MAP in a liquid matrix, although some MAP survival on some occasions was observed.

A sustained release of anti-infection drugs is beneficial for the reduction of required dressing change times and the protection of the granulation tissue against further infection. In this work, the strategy of encapsulating mesoporous SBA-15 particles in a permeable cellulose membrane (CM) was used in the preparation of antibacterial cellulose based composite membranes to depot store and deliver drugs with prolonged anti-microbial drug release characteristics. It was found that SBA-15 with the protection of phosphate buffered saline prefilled in SBA-15 mesoporous channel could resist strong alkali environment during composite membranes preparation and its mesoporous structure remained intact, which was verified by SEM, TEM and BET results. The resultant cellulose based composite membrane containing 30 wt% SBA-15 (denoted as P-CM-SBA (30%)) had achieved 3.6 wt% drug loading and demonstrated the sustained release of chloramphenicol for 250 h, which was resulted from SBA-15/cellulose structure-inducing two-stage release behavior. Strong antibacterial activity of P-CM-SBA (30%) against S. aureus and E. coli. could last 144 h. In addition, the tensile strength, water vapor transmission rate and swelling properties of P-CM-SBA (30%) conformed to the primary requirements of wound dressing materials. Therefore, cellulose based composite membrane with 30 wt% SBA-15 particles possesses the potential for the application of wound healing. [ABSTRACT FROM AUTHOR]

Abstract Background & aim: Easy access, rapid recovery and high potency of monocyte cell therapy have led to special attention in cell therapy research. Monocytes are considered as sticky cells to the flask. Therefore, finding the appropriate isolation method that has the least damage to the cell and its function is of particular importance. The purpose of this study was to compare the functional capabilities of monocytes after isolation with three methods: lidocaine / EDTA, trypsin and cold PBS / EDTA. Methods: In this experimental study, after extraction of peripheral blood mononuclear cells from Balb / c mice, cells (107 × 1 cells / ml) were incubated in RPMI culture medium in T25 culture flask for 4 hours. After incubation time, non-adherent cells (mainly lymphocytes) were separated by two rinsing and removed from the flask. Three different methods of trypsin, lidocaine and phosphate buffer saline were used for isolation of monocyte cells. After isolating the cells with each method, the functional capabilities of the monocytes were measured and compared with each other. The collected data were analyzed using one-way ANOVA. Results: The amount of extraction, survival, metabolic activity, phagocytosis percentage, respiratory explosion, nitric oxide levels, and yeast potential in cells isolated by lidocaine were significantly higher than other groups. Although the use of trypsin, although it results in the removal of more cells, is cooled to the PBS method, but these cells exhibit significant physiological impairment in comparison with the Lidocaine / EDTA or PBS / EDTA method. Neutral red uptake was detected by trypsin isolated monocyte cells in comparison to the other two methods at lower levels. In comparison between cold PBS and lidocaine, it seems that there is no significant difference between the monocytes obtained from the two methods in terms of neutralization. Conclusion: Compared to trypsin and PBS / EDTA, the Lidocaine / EDTA method is an appropriate method for isolating monocytes adherent to the flask, due to the extraction of more efficient and efficient cells. .

Staphylococcus aureus and Mycobacterium tuberculosis are major causative agents responsible for serious nosocomial and community-acquired infections impacting healthcare systems globally. Over several decades, these pathogens have developed resistance to multiple antibiotics significantly affecting morbidity and mortality. Thus, these recalcitrant pathogens are amongst the most formidable microbial pathogens for which international healthcare agencies have mandated active identification and development of new antibacterial agents for chemotherapeutic intervention. In our present work, a series of new quinazolin-4(3 H)-one derivatives were designed, synthesized and evaluated for their antibacterial activity against ESKAP pathogens and pathogenic mycobacteria. The experiments revealed that 4'c , 4'e , 4'f and 4'h displayed selective and potent inhibitory activity against Staphylococcus aureus with MIC values ranging from 0.03-0.25 μ g/mL. Furthermore, compounds 4'c and 4'e were found to be benign to Vero cells (CC 50 = >5 μ g/mL) and displayed promising selectivity index (SI) > 167 and > 83.4 respectively. Additionally, 4'c and 4'e demonstrated equipotent MIC against multiple drug-resistant strains of S. aureus including VRSA, concentration dependent bactericidal activity against S. aureus and synergized with FDA approved drugs. Moreover, compound 4′c exhibited more potent activity in reducing the biofilm and exhibited a PAE of ∼2 h at 10X MIC which is comparable to levofloxacin and vancomycin. In vivo efficacy of 4'c in murine neutropenic thigh infection model revealed that 4'c caused a similar reduction in cfu as vancomycin. Gratifyingly, compounds 4d , 4e , 9a , 9b , 14a , 4'e and 4'f also exhibited anti-mycobacterial activity with MIC values in the range of 2-16 μ g/mL. In addition, the compounds were found to be less toxic to Vero cells (CC 50 = 12.5->100 μ g/mL), thus displaying a favourable selectivity index. The interesting results obtained here suggest the potential utilization of these new quinazolin-4(3 H)-one derivatives as promising antibacterial agents for treating MDR-Staphylococcal and mycobacterial infections. Image 1 • Series of new quinazolin-4(3 H)-one derivatives were designed, synthesized and evaluated for antibacterial activity. • 4'c , 4'e , 4'f and 4'h displayed selective and potent inhibitory activity against Staphylococcus aureus. • 4'c and 4'e were found to be non-toxic to Vero cells and displayed promising SI. • 4'c and 4'e demonstrated equipotent MIC against multiple drug-resistant strains of S. aureus , concentration dependent bactericidal activity against S. aureus and synergized with tested drugs. • 4′c exhibited more potent activity in reducing the biofilm and exhibited a PAE of ∼2 h at 10X MIC and also exhibited similar in vivo efficacy as Vancomycin. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) primary cells can be isolated from peripheral blood, suspended with media containing bovine serum and cryoprotectant, and stored in liquid nitrogen before being processed for proteomic analysis by mass spectrometry (MS). The presence of bovine serum and human blood proteins in AML samples can hamper the identifications of proteins, and thereby reduce the proteome coverage of the study. Herein, we have established the effect of phosphate buffered saline (PBS) washing on AML patient samples stored in media. Although PBS washes effectively removed serum and blood contaminants, the saline wash resulted in cell burst and remarkable protein material loss. We also compared different methods to preserve the AML proteome from THP-1 and Molm-13 cell lines before MS analysis: (1) stored in media containing bovine serum and dimethyl sulfoxide (DMSO); (2) stored as dried cell pellets; and (3) stored as cell lysates in 4% sodium dodecyl sulfate (SDS). MS analysis of differently preserved AML cell samples shows that preservation with DMSO produce a high number of fragile cells that will burst during freezing and thawing. Our studies encourage the use of alternative preservation methods for future MS analysis of the AML proteome. [ABSTRACT FROM AUTHOR]

ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. This new approach is easy to perform and adaptable in most laboratories. In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. coli , Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates. Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes. [ABSTRACT FROM AUTHOR]

The electrochemical reduction of graphene oxide (GO) is an environmentally friendly and energy-saving method for improving the characteristics of GO. However, GO films must be coated on the cathode electrode in advance when usingthis technique. Thus, the formed electrochemically reduced GO (ERGO) films can be used only as sensing or conductive materials in devices because mass production of the flakes is not possible. Therefore, this study proposes a facile electrochemical reduction technique. In this technique, GO flakes can be directly used as reduced materials, and no GO films are required in advance. A 0.1 M phosphate buffered saline solution was used as the electrolyte, which is a highly safe chemical agent. Experimental results revealed that the as-prepared GO flakes were electrochemically reduced to form ERGO flakes by using a −10 V bias for 8 h. The ratio of the D-band and G-band feature peaks was increased from 0.86 to 1.12, as revealed by Raman spectroscopy, the π-π stacking interaction operating between the ERGO and GO has been revealed by UV-Vis absorption spectroscopy, and the C–O ratio was increased from 2.02 to 2.56, as indicated by X-ray photoelectron spectroscopy. The electrical conductivity of the ERGO film (3.83 × 10−1 S·cm−1) was considerably better than that of the GO film (7.92 × 10−4 S·cm−1). These results demonstrate that the proposed electrochemical reduction technique can provide high-quality ERGO flakes and that it has potential for large-scale production.

Biodegradable magnesium alloys including AZ31 are exciting candidates for temporary implants as they eliminate the requirement for surgical removal, yet have higher mechanical properties than degradable polymers. However, the very long term mechanical properties and degradation of these alloys have not been fully characterized. The tensile, bending and corrosion behaviour of biodegradable AZ31 Mg alloy specimens have been investigated for up to 9 months in vitro in phosphate buffered saline (PBS). Small AZ31 Mg specimens showed a significant drop in bend yield strength and modulus after 3 months in vitro degradation and an average mass loss of 6.1%. Larger dumbbell specimens showed significant drops in tensile strength from 251.96 ± 3.53 MPa to 73.5 ± 20.2 MPa and to 6.43 ± 0.9 MPa and in modulus from 47.8 ± 5.6GPa to 25.01 ± 3.4GPa and 2.36 ± 0.89GPa after 3 and 9 months respectively. These reductions were accompanied by an average mass loss of 18.3% in 9 months. Degradation rate for the small and large specimens followed similar profiles with immersion time, with peak degradation rates of 0.1747 g m − 2 h − 1 and 0.0881 g m − 2 h − 1 , and average rates of 0.1038 g m − 2 h − 1 and 0.0397 g m − 2 h − 1 respectively. SEM fractography and polished specimen cross-sections revealed corrosion pits, cracks and corrosion induced defects. These data indicate the potential of AZ31 Mg for use in implants that require medium term degradation with load bearing mechanical properties. [ABSTRACT FROM AUTHOR]

Background: Toxoplasmosis is considered as one of the most common infectious diseases caused by the protozoan parasite Toxoplasma gondii. Tachyzoite is the main form of Toxoplasma and continuously is maintained in cell culture or injected into the mice peritoneal cavity. This study was designed to evaluate the survival rate of RH strain of T. gondii tachyzoites in different cell free, nutrient and biological media at different temperatures. Methods: This experimental study was performed at the Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari, Iran, in 2010. One ml of each solution including hypotonic saline (0.3%), normal saline (0.85%), RPMI-1640 (RPMI), RPMI with 10% fetal bovine serum (FBS), RPMI with 20% FBS, ovine hydatid cyst fluid, pasteurized milk of cow, and phosphate buffered saline (PBS) along with 4×104 T. gondii tachyzoites were added to plate wells and incubated in 4 °C, 22 °C, 37 °C, and 37 °C under 5% CO2. The survival rate and viability as­sessment of parasites were performed daily and the results were analyzed using Univariate tests. Result: Tachyzoites survival rate in PBS (4 °C) and normal saline (4 °C) were con­siderably high, compared to other solutions in different conditions (P

Background: Toxoplasmosis is considered as one of the most common infectious diseases caused by the protozoan parasite Toxoplasma gondii. Tachyzoite is the main form of Toxoplasma and continuously is maintained in cell culture or injected into the mice peritoneal cavity. This study was designed to evaluate the survival rate of RH strain of T. gondii tachyzoites in different cell free, nutrient and biological media at different temperatures. Methods: This experimental study was performed at the Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari, Iran, in 2010. One ml of each solution including hypotonic saline (0.3%), normal saline (0.85%), RPMI-1640 (RPMI), RPMI with 10% fetal bovine serum (FBS), RPMI with 20% FBS, ovine hydatid cyst fluid, pasteurized milk of cow, and phosphate buffered saline (PBS) along with 4×104 T. gondii tachyzoites were added to plate wells and incubated in 4 °C, 22 °C, 37 °C, and 37 °C under 5% CO2. The survival rate and viability assessment of parasites were performed daily and the results were analyzed using Univariate tests. Result: Tachyzoites survival rate in PBS (4 °C) and normal saline (4 °C) were considerably high, compared to other solutions in different conditions (P<0.001). The best temperature for Toxoplasma maintenance was 4 °C (P<0.001). Conclusion: This study introduces two available and economical solutions, PBS (4 °C) and normal saline (4 °C) media, for maintenance of Toxoplasma tachyzoites as appropriate choice media for a noticeable period of time (11 days) in vitro. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNPs) have been used for decades as anti-bacterial agents in various industrial fields such as cosmetics, health industry, food storage, textile coatings and environmental applications, although their toxicity is not fully recognized yet. Antimicrobial and catalytic activity of AgNPs depends on their size as well as structure, shape, size distribution, and physico-chemical environment. The unique properties of AgNPs require novel or modified toxicological methods for evaluation of their toxic potential combined with robust analytical methods for characterization of nanoparticles applied in relevant vehicles, e.g., culture medium with/without serum and phosphate buffered saline. [ABSTRACT FROM AUTHOR]

As research progresses to find a suitable knee meniscus replacement, accurate in vitro testing becomes critical for feasibility and comparison studies of mechanical integrity. Within the knee, the meniscus is bathed in synovial fluid, yet the most common hydration fluid in laboratory testing is phosphate buffered saline (PBS). PBS is a relatively simple salt solution, while synovial fluid is a complex non-Newtonian fluid with multiple lubricating factors. As such, PBS may interact with meniscal tissue differently than synovial fluid, and thus, the hydration fluid may be an important factor in obtaining accurate results during in vitro testing. To evaluate these effects, medial porcine menisci were used to evaluate tissue mechanics in tension ( n =11) and compression ( n =15). In all tests, two samples from the same meniscus were taken, where one sample was hydrated in PBS and the other was hydrated in synovial fluid. Statistical analysis revealed no significant differences between the mean mechanical properties of samples tested in PBS compared to synovial fluid; however, compressive testing revealed the variability between samples was significantly reduced if samples were tested in synovial fluid. For example, the compressive Young׳s Modulus was 12.69±7.49 MPa in PBS versus 12.34±4.27 MPa in synovial fluid. These results indicate testing meniscal tissue in PBS will largely not affect the mean value of the mechanical properties, but performing compression testing in synovial fluid may provide more consistent results between samples and assist in reducing sample numbers in some experiments. [ABSTRACT FROM AUTHOR]

We herein report the spontaneous interfacial reaction between copper foil with 0.01 M phosphate buffered saline (PBS) to form free-standing cupric phosphate (Cu 3 (PO 4 ) 2 ) nanoflowers at ambient temperature. The underlying chemistry was thoroughly investigated and it is found that the formation of nanoflower is synergistically caused by dissolved oxygen, chlorine ions and phosphate ions. Enzyme-Cu 3 (PO 4 ) 2 hybrid nanoflower was further prepared successfully by using an enzyme-dissolving PBS solution and the enzymes in the hybrid exhibit enhanced biological activity. This work provides a facile route for large-scale synthesis of hierarchical inorganic and functional protein-inorganic hybrid architectures via a simple one-step solution-immersion reaction without using either template or surfactant, thus offering great potential for biosensing application among others. [ABSTRACT FROM AUTHOR]

A novel bacterial cellulose (BC) membrane supported silver nanoparticles (AgNPs) was synthesized with a facile in situ growth method. The physicochemical properties of AgNP/BC were characterized by scanning electron microscope (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD). The results demonstrate that AgNPs with diameters of 10–20 nm were anchored throughout the three-dimensional network of BC membrane. The as-prepared AgNP/BC membrane was used as cathodic electrode to investigate its catalytic activity to oxygen reduction reaction (ORR) in phosphate buffered saline (PBS) for the first time. As a result, the maximum current density obtained with AgNP/BC was −3.94 mA cm −2 , which was higher than Ag electrode and AgNP. Besides, the maximum current density of AgNP/BC only decreased 4.3% in electrolyte with 10 mmol L −1 glucose. Such AgNP/BC membrane with advantages including biocompatibility, facile synthesis, high catalytic activity and good glucose tolerance could be valuable to ORR in implanted glucose fuel cells. [ABSTRACT FROM AUTHOR]

Structure of hydroxyapatite (HAP) is more flexible towards ionic substitutions. Properties such as solubility, antimicrobial property can be tailored by substitutions. Substituted hydroxyapatite and fluorapatite of formulae BiNaCa 3 (PO 4 ) 3 OH, Bi 0.5 M 0.5 Ca 4 (PO 4 ) 3 OH (M=K, Ag), Ca 10− x Cu x (PO 4 ) 6 (OH/F) 2 d( x =0.05–0.25) and Bi 0.5 Na 0.5 Ca 4 (PO 4 ) 3 F were synthesized and characterized by powder XRD, FT-IR, SEM–EDAX and TGA. In vitro solubility of the synthesized compounds was studied in the phosphate buffered medium of pH 7.4 at 37 °C. Based on the release of calcium and phosphorus ion concentration and pH, the solubility of these compounds is discussed. Bismuth and sodium co-substituted hydroxyapatite are found to be more soluble compared with other substituted apatite compounds and unsubstituted hydroxyapatite. [ABSTRACT FROM AUTHOR]

In the search for effective therapeutic strategies, protein-based biologicals are under intense development. While monoclonal antibodies represent the majority of these drugs, other innovative approaches are exploring the use of scaffold proteins for the creation of binding molecules with tailor-made properties. Ubiquitin is especially suited for this strategy due to several key characteristics. Ubiquitin is a natural serum protein, 100% conserved across the mammalian class and possesses high thermal, structural and proteolytic stability. Because of its small size and lack of posttranslational modifications, it can be easily produced in Escherichia coli . In this work we provide evidence that ubiquitin is safe as tested experimentally in vivo . In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4. Cellular assays based on different signaling pathways of the receptor were conducted with the natural agonist SDF-1 as a benchmark. In none of the assays could a response to ubiquitin treatment be elicited. Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters. [ABSTRACT FROM AUTHOR]

Cosic discovered that spectral analyses of a protein sequence after each constituent amino acid had been transformed into an appropriate pseudopotential predicted a resonant energy between interacting molecules. Several experimental studies have verified the predicted peak wavelength of photons within the visible or near-visible light band for specific molecules. Here, this concept has been applied to a classic signaling pathway, JAK–STAT, traditionally composed of nine sequential protein interactions. The weighted linear average of the spectral power density (SPD) profiles of each of the eight “precursor” proteins displayed remarkable congruence with the SPD profile of the terminal molecule (CASP-9) in the pathway. These results suggest that classic and complex signaling pathways in cells can also be expressed as combinations of resonance energies. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β-amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and β C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing. [ABSTRACT FROM AUTHOR]

Insufficient supply of manufacture's buffers/diluents in relation to the number of strips per kit has been found to have negative impact on patients' results. Some laboratories personnel tend to use diluents from other rapid tests manufacturers such as Bioline, Unigold as well as malaria rapid diagnostic test (MRDT). This study aimed at evaluating the use of 0.01M phosphate buffered saline (PBS) as detection buffer for Alere Determine® HIV rapid test. This study was carried out at Bugando School of Medicine in Mwanza, Tanzania. A total of 300 whole blood specimens; 150 HIV positive specimens from patients attending Care and Treatment Centreand 150 HIV negative specimens were retested for HIV status using Alere Determine® HIV rapid test employing normal Alere buffer and 0.01M PBS as buffer.Of the total specimens tested; 150 (100%) of HIV positive were positive by using both Alere buffer and 0.01M PBS while 150(100%) of HIV negative samples were negative by both Alere Determine® and 0.01M PBS. The agreement between 0.01M PBS and Alere Determine® buffer was 100%. The value of kappa indicates perfect agreement between 0.01M PBS and Alere Determine® buffer (100%). A 0.01M PBS is recommended as alternative detection buffer for Alere Determine® in cases of insufficient supply. Further investigation to evaluate the suitable buffer for other rapid tests for HIV and other diseases is recommended especially in resource limited settings. [ABSTRACT FROM AUTHOR]

Loss of endothelial adherens junctions is involved in tumor metastasis. Here, we demonstrate that, in the metastatic Lu1205 melanoma cells, expression of the CD44 variant CD44v8‐v10 induced junction disassembly and vascular endothelial (VE)‐cadherin phosphorylation at Y658 and Y731. Short interfering RNA (siRNA)‐mediated CD44 knockdown or sialic acid cleavage reversed these effects. Moreover, microspheres coated with recombinant CD44v8‐v10 promoted endothelial junction disruption. Overexpression of CD44v8‐v10 but not of standard CD44 (CD44s) promoted gap formation in the non‐metastatic WM35 melanoma cells, whereas CD44 knockdown or neuraminidase treatment dramatically diminished melanoma transendothelial migration. Endothelial cells transfected with the phosphomimetic VE‐cadherin mutant Y658E supported transmigration of CD44‐silenced Lu1205 cells. Our findings imply that CD44 variant isoform (CD44v) but not CD44s regulates endothelial junction loss, promoting melanoma extravasation.Melanoma contact induced VE‐cadherin phosphorylation and endothelial gap formation. Lu1205 melanoma cell expresses high levels of CD44v8‐v10. CD44v8‐v10 but not CD44s facilitated Lu1205‐induced endothelial gap formation. CD44v8‐v10 overexpression enhanced the ability of WM35 to induce junction loss. CD44 knockdown suppressed VE‐cadherin disassembly‐dependent Lu1205 extravasation. [ABSTRACT FROM AUTHOR]

Formation of bacterial biofilms at solid–liquid interfaces creates numerous problems in biomedical sciences. Conventional sterilization and decontamination methods are not suitable for new and more sophisticated biomaterials. In this paper, the efficiency and effectiveness of gas discharges in the inactivation and removal of biofilms on biomaterials were studied. It was found that although discharge oxygen, nitrogen and argon all demonstrated excellent antibacterial and antibiofilm activity, gases with distinct chemical/physical properties underwent different mechanisms of action. Discharge oxygen- and nitrogen-mediated decontamination was associated with strong etching effects, which can cause live bacteria to relocate thus spreading contamination. On the contrary, although discharge argon at low powers maintained excellent antibacterial ability, it had negligible etching effects. Based on these results, an effective decontamination approach using discharge argon was established in which bacteria and biofilms were killedin situand then removed from the contaminated biomaterials. This novel procedure is applicable for a wide range of biomaterials and biomedical devices in anin vivoand clinical setting. [ABSTRACT FROM PUBLISHER]

Cis-diamminedichloroplatinum(II) (cisplatin) was immobilized in a star-shaped polymer carrier. In order to define whether this polymer could be used as a drug delivery system for cisplatin, its characteristics, e.g. loading efficiency and release profile of cisplatin from the polymer in phosphate buffered saline (PBS), have to be assessed, and hence an appropriate analytical procedure for precise and reliable determination of Pt(II) must be established. Optimal instrumental parameters for Pt measurement by commonly used atomic spectrometric methods ETAAS and ICP-OES in various examined media were defined. Matrix interferences observed that were related to both techniques were evaluated, and analytical figures of merit were compared and critically discussed. Graphical abstract: [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The antibacterial peptide Cecropin B (CB), isolated from the giant silk moth, has been shown to effectively eliminate bacteria. In this study, the effects of transgenic CB on dairy goat mammary epithelial cells (DGMECs) and dairy goat mammary gland were investigated. The DNA of CB from silkworm was amplified by reverse transcription PCR (RT-PCR) and then fused to the eukaryotic expression vector pECFP-C1. The recombinant plasmid pECFP-Cecropin B (pECFP-CB) was used for the transfection of DGMECs, and the expression of transgenic CB and the antibacterial activity of it were confirmed by western blot and agar diffusion reaction respectively. The stable DGMEC line transfected by pECFP-CB was obtained by screening with G418. In vivo experiment, pECFP-CB was injected into dairy goat mammary gland, and also the expression and antibacterial activity of transgenic CB were confirmed. Results of this study: transgenic CB can be expressed in DGMECs and dairy goat mammary gland, and inhibit the mastitis caused byStaphylococcus aureus. [ABSTRACT FROM PUBLISHER]

Abstract: In order to be used as a material for neural prostheses, polyimide has to withstand the body environment over years. To obtain more information about the long-term stability of this material, we tested three commercially available polyimides (PI2611 – HD-Microsystems (type: BPDA-PPD), U-Varnish-S – UBE (type: BPDA-PPD), Durimide 7510 – Fujifilm (type: information not provided)). Specimens were stored in phosphate buffered saline (PBS) at 37 °C to simulate body temperature and at elevated temperatures of 60 °C and 85 °C to accelerate aging. During the course of 20 months, stress-strain curves were measured monthly by tensile testing. From these curves the Young''s modulus, the stress and strain at break, the stress at 10% strain as well as the fracture energy were extracted and used to characterize and to statistically evaluate the mechanical material properties. Mass loss was determined by weighing polyimide foils (Upilex25S - UBE) and optical measurements were conducted to examine changes in chemical structure and crystallinity. At 37 °C and 60 °C no change in material behavior could be observed, except for an increase of the Young''s modulus of the BPDA-PPD type stored at 60 °C. This demonstrates the long-term stability of all tested polyimides with respect to PBS. All extracted quantities mentioned above, as well as the mass, decreased in specimens stored in PBS at 85 °C. As a comparison, BPDA-PPD type specimens stored at 85 °C in deionized water showed no change in any property but behaved similarly to the reference material. [Copyright &y& Elsevier]

Abstract: African trypanosomes are unable to synthesize purines de novo and must salvage preformed purine nucleosides and nucleobases from their hosts. The Trypanosoma brucei genome project has identified 12 members of the equilibrative nucleoside transporter family, most of which have been characterized previously as nucleoside and/or nucleobase transporters. Here the 11th member of this family, TbNT11.1, has been functionally expressed in null mutants of Leishmania that are deficient in purine nucleoside or nucleobase uptake and identified as a high-affinity purine nucleobase transporter. Expression of TbNT11.1 in Xenopus oocytes revealed that it is also a transporter for the diamidine drug pentamidine that is the principal drug employed to treat early stage human African trypanosomiasis and may thus contribute to the uptake of this therapeutically important compound. In addition, characterization of the 12th member of the family, TbNT12.1, reveals that it is an adenine/pentamidine transporter. [Copyright &y& Elsevier]

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by gait dysfunction in later stages of the disease. PD hallmarks include a decrease in stride length, run speed, and swing time; an increase in stride time, stance time, and base of support; dopaminergic degeneration in the basal ganglia; and cholinergic degeneration in the pedunculopontine nucleus (PPN). A progressive animal model of PD is needed to identify treatments for gait dysfunction. The goal of this study was to quantify progressive gait degeneration in PTEN-induced putative kinase 1 knockout (P1KO) rats and investigate neurodegeneration as potential underlying mechanisms. Gait analysis was performed in male P1KO and wild-type rats at 5 and 8 months of age and immunohistochemical analysis at 8 months. Multiple parameters of volitional gait were measured using a runway system. P1KO rats exhibited significant gait deficits at 5 months, but not 8 months. Gait abnormalities improved over time suggesting compensation during behavioral testing. At 8 months a 15% loss of tyrosine hydroxylase (TH) in the striatum, a 27% loss of TH-positive cells in the substantia nigra pars compacta, and no significant loss of choline acetyltransferase-positive cells in the PPN was found. Dopaminergic cell loss may contribute to gait deficits in the P1KO model, but not cholinergic cell loss. The P1KO rat with the greatest dopamine loss exhibited the most pronounced PD-like gait deficits, highlighting variability within the model. Further analysis is required to determine the suitability of the P1KO rat as a progressive model of gait abnormalities in PD. [ABSTRACT FROM AUTHOR]

Abstract: The epidemiological studies and recent data have provided convinced evidence that green tea and its major constituent epigallocatechin gallate (EGCG) might have the potential to lower the risk of cancers in humans. Metal ions, such as zinc and cadmium, which are necessary to our health, are important factors inducing many diseases including prostate cancer in the condition of absence or excess. EGCG can satisfactorily exhibit complex chemistry with metal ions because of multiple hydroxyl states, which in turn changes their bioactivities and metabolism pathways. This paper presents the results of an investigation of the cytotoxicity of EGCG against PC-3 prostate cancer cells in the presence and absence of Cd2+ in vitro. The results showed that both EGCG and Cd2+ suppressed viability and clonegenecity of PC-3 cells, and the suppression effect was enhanced when EGCG added with Cd2+. Although Cd2+ up-regulated the 67kDa laminin receptor (67LR), which is a migration-associated protein, the cell migration ability was not significantly increased after each treatment. We also found that EGCG and Cd2+ directly interacted with mitochondrial, and the mixture of EGCG and Cd2+ (EGCG+Cd2+) significantly caused loss of the mitochondrial membrane potential, decrease of the ATP content and activation of caspase-9 compared with EGCG treated alone. Taken together, these findings suggest that Cd2+ enhanced the cytotoxicity of EGCG to PC-3 cells by up-regulating the 67LR and the mitochondria-mediated apoptosis pathway. [Copyright &y& Elsevier]

Abstract: This study reports the first comprehensive data on the effects of glycerol, a widely used cryoprotective agent (CPA), on the thermal properties of Phosphate Buffered Saline at subzero and cryogenic (⩽−40°C) temperatures. A Differential Scanning Calorimeter was used to measure specific and latent heats. Thermal conductivity was determined using a thermistor probe technique. Results differed significantly from known water (ice) or glycerol values, and a simple weight averaging method between water and glycerol was found to over-predict thermal conductivity below −100°C due to glass formation. This indicates the need for these newly measured thermal properties of CPA solutions particularly below −100°C. [Copyright &y& Elsevier]

Abstract: Fibrin deposition is a hallmark of pleural inflammation and loculation but understanding of mechanisms by which mesothelial cells regulate intrapleural fibrinolysins remains incomplete. We speculated that pleural mesothelial cells regulate local fibrinolytic capacity via processing of single chain urokinase type plasminogen activator (scuPA). Pretreatment of human pleural mesothelial (MeT-5A) cells with TGF-β or thrombin, either alone or in combination, inhibited urokinase (uPA)-mediated fibrinolysis by MeT-5A. Thrombin, unlike TGF-β, inhibited fibrinolysis without induction of PAI-1, suggesting that thrombin-mediated cleavage of scuPA inhibits the fibrinolytic capacity of MeT-5A cells. Thrombin cleaves both purified scuPA as well as that secreted by MeT-5A cells and cell surface thrombomodulin accelerates thrombin-mediated cleavage of scuPA to inhibit cellular fibrinolytic activity. Molecular dynamics analyses demonstrated that thrombin-cleaved scuPA (uPAt) do not acquire a catalytically active conformation and that secondary plasminogen binding sites of uPA implicated in plasminogen activation are distorted in uPAt, explaining, at least in part, why uPAt is a poor enzyme. uPAt was detectable in transudative and exudative pleural effusions from patients. Intrapleural administration of scuPA generated increased levels of uPAt in PF of rabbits with pleural injury and loculation induced by tetracycline in vivo. This pathway is operative in diverse forms of pleural injury, restricts the urokinase-dependent fibrinolytic capacity of pleural mesothelial cells and contributes to local control of fibrinolytic activity via processing of endogenous or exogenous scuPA within the pleural compartment. [Copyright &y& Elsevier]

Abstract: Escherichia coli O157:H7 is a serious human pathogen that causes hemorrhagic colitis, and occasionally hemolytic uremic syndrome. Identification of the O157 antigen is an essential part of the detection and management of E. coli O157:H7. A quadroma P126 secreting a bispecific hybrid MAb (bsMAb), which recognizes both E. coli O157 and horseradish peroxidase in one molecule was produced by somatic hybridization of hybridomas specific for E. coli O157 and HRPO molecule. A bridge ELISA was used to select the quadromas obtained for bispecific monoclonal antibody purification and characterization. Benzhydroxamic-acid agarose (BHA) affinity co-chromatography was used as a convenient one-step method for purifying the HRPO–bsMAb complex for ultrasensitive diagnostic applications. Sandwich ELISA for detecting E. coli O157:H7 with HRPO–bsMAb allows quick one step detection of spiked E. coli O157:H7. The detection sensitivities were 100 CFU, 750 CFU and 500 CFU per 1 ml of tap water, lake water and apple juice respectively by microtiter assay. E. coli O157:H7 detection with immunofilter ELISA and immunomagnetic ELISA formats was approximately 1 CFU/ml and 10 CFU/ml respectively. BsMAbs avoid enzyme conjugation, has highest specific activity and molecular uniformity without aggregates and contribute to good signal to noise ratios. This new bispecific antibody can be generated and purified from quadroma cultures by affinity co-chromatography in one step and can be used to develop a new generation of assays for public health applications in water, food and human sample testing. [Copyright &y& Elsevier]

Abstract: The objective of this work is to identify proteins of the human and porcine parasite, Taenia solium, which may be exploited for control of the parasite. Through screening a cDNA library of T. solium metacestodes, we have identified a novel Sec-14-like Taenia lipid-binding protein that may play an important role in membrane trafficking. The Sec14-like sequence is a single copy gene, encoding a putative polypeptide of 320 amino acids and 36.1 kDa (sec14Tsol protein). Secondary amino acid structural analysis suggested that the sec14Tsol protein might contain two distinct structural domains, an amino-terminal α-helix rich domain and a mixed α-helix/β-stand carboxy-terminal zone, showing homology with the conserved SEC14 domain found in a great number of proteins that bind lipids, as the regulators of membrane trafficking between Golgi membrane bilayers. Significantly, therefore, in a phosphoinositide-binding assay, sec14Tsol purified recombinant protein specifically interacted with important lipid regulators of membrane trafficking, with a preference for PI(3)P2, PI(3,4)P2, PI(4,5)P2 and phosphatidic acid. Moreover, the sec14Tsol protein was localized in the Golgi apparatus of transfected cells and in the spiral canal region of T. solium metacestode tegument. As sec14Tsol protein may play an important role in membrane trafficking, its demonstrated localisation in the intact parasite tegument suggests its involvement in the function of the tegument and thus perhaps interaction with the host. [Copyright &y& Elsevier]

Abstract: Peptidoglycan recognition protein (PGRP) was isolated from immunized hemolymph of the wild silkworm, Samia cynthia ricini, detecting the biding activity with 125I-labeled peptidoglycan (PGN). The binding specificity of PGRP was tested by competitive inhibition of the binding to 125I-labeled-PGN by a large excess amount of non-labeled-PGN or other glucans. The binding to labeled uncross-linked Lys-type PGN from Micrococcus luteus was strongly inhibited by non-labeled-PGN of the same structure and meso-diaminopimelic acid (DAP)-type cross-linked PGN from Bacillus cell wall, but only a little by cross-linked PGN from M. luteus cell wall. The PGRP cDNA encodes a 193 amino acid open reading frame. The deduced amino acid sequence had 62 to 91% identities to known lepidopteran PGRPs, but less than 40% to Drosophila PGRPs. The PGRP gene constitutively expressed at a low level in naive fat body, and strongly induced by an injection of DAP-type cross-linked and Lys-type uncross-linked PGNs, but only weakly by Lys-type cross-linked PGN from M. luteus. The silkworm possibly distinguish between PGNs based on the structure of cross-linking peptide, but has less if any preference for the diamino acid residue of the stem peptide. [Copyright &y& Elsevier]

Abstract: Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k a and k d) and affinity (K D) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54×10−8 to 3.53×10−10 M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day. [Copyright &y& Elsevier]

Abstract: Glucose is a major source of energy and carbon in promastigotes of Leishmania mexicana, and its uptake is mediated by three glucose transporters whose genes are encoded within a single cluster. A null mutant in which the glucose transporter gene cluster was deleted by homologous gene replacement was generated previously and shown to grow more slowly than wild type promastigotes but not to be viable as amastigotes in primary tissue culture macrophages or in axenic culture. Further phenotypic characterization demonstrates that the null mutant is unable to import glucose, mannose, fructose, or galactose and that each of the three glucose transporter isoforms, LmGT1, LmGT2, and LmGT3, is capable of transporting each of these hexoses. Complementation of the null mutant with each isoform is able to restore growth in each of the four hexoses to wild type levels. Null mutant promastigotes are reduced in size to about 2/3 the volume of wild type parasites. In addition, the null mutants are significantly more sensitive to oxidative stress than their wild type counterparts. These results underscore the importance of glucose transporters in the parasite life cycle and suggest reasons for their non-viability in the disease-causing amastigote stage. [Copyright &y& Elsevier]

Abstract: To examine the effects of increased expression of Cx50 in the mouse lens, transgenic mice were generated using a DNA construct containing the human Cx50 coding region and a C-terminal FLAG epitope driven by the chicken βB1-crystallin promoter. Expression of this protein in paired Xenopus oocytes induced gap junctional currents of similar magnitude to wild type human Cx50. Three lines of transgenic mice expressing the transgenic protein were analyzed. Lenses from transgenic mice were smaller than those from non-transgenic littermates, and had cataracts that were already visible at postnatal day 1. Expression of the transgene resulted in a 3- to 13-fold increase in Cx50 protein levels above those of non-transgenic animals. Light microscopy revealed alterations in epithelial cell differentiation, fiber cell structure, interactions between fiber cells and areas of liquefaction. Scanning electron microscopy showed fiber cells of varying widths with bulging areas along single fibers. Anti-Cx50 and anti-FLAG immunoreactivities were detected at appositional membranes and in intracellular vesicles in transgenic lenses. N-cadherin, Cx46, ZO-1 and aquaporin 0 localized mainly at the plasma membrane, although some N-cadherin and aquaporin 0 was associated with the intracellular vesicles. The abundance and solubility/integrity of αA-, αB-, β- and γ-crystallin were unaffected. These results demonstrate that transgenic expression of Cx50 in mice leads to cataracts associated with formation of cytoplasmic vesicles containing Cx50 and decreased or slowed epithelial differentiation without major alterations in the distribution of other integral membrane or membrane-associated proteins or the integrity/solubility of crystallins. [Copyright &y& Elsevier]

Objective: Superficial zone protein (SZP) has been shown to function in the boundary lubrication of articular cartilages of the extremities. However, the expression of SZP has not been clarified in mandibular cartilage which is a tissue that includes a thick fibrous layer on the surface. This study was conducted to clarify the distribution of SZP on the mandibular condyle and the regulatory effects of humoral factors on the expression in both explants and fibroblasts derived from mandibular condyle.Methods: The distribution of SZP was determined in bovine mandibular condyle cartilage, and the effects of interleukin-1beta (IL-1beta) and transforming growth factor-beta (TGF-beta) on SZP expression were examined in condyle explants and fibroblasts derived from the fibrous zone of condyle cartilage.Results: SZP was highly distributed in the superficial zone of intact condyle cartilage. The SZP expression was up-regulated by TGF-beta in both explants and cultured fibroblasts, whereas the expression was slightly down-regulated by IL-1beta. A significant increase in accumulation of SZP protein was also observed in the culture medium of the fibroblasts treated with TGF-beta.Conclusions: These results suggest that SZP plays an important role in boundary lubrication of mandible condylar cartilage, is synthesized locally within the condyle itself, and exhibits differential regulation by cell mediators relevant to mandibular condyle repairing and pathologies. [ABSTRACT FROM AUTHOR]