In vitro Assessment of Tris Egg Yolk and Soybean Lecithin Based Extenders for Cryopreservation of Crossbred Ram Semen.

BACKGROUND: The replacement of egg yolk with alternative plant-derived soybean lecithin is gaining interest in both animal and human sperm cryopreservation owing to biosecurity issues with egg yolk based extenders. OBJECTIVE: To evaluate the comparative effect of egg yolk and soyabean lecithin based extenders on the quality of cryopreserved crossbred ram semen. METHODS: Pooled ejaculates (total ejaculates = 36) were divided into two aliquots and extended with Tris egg yolk extender (Tris extender) and soybean lecithin based commercial extender (Ovixcell) RESULTS: Among the two extenders, Ovixcell showed better sperm quality both at the pre-freeze (Sperm motility) and post-thaw stages. Lower malondialdehyde (MDA) level (nmol/mL) was observed in Ovixcell as compared to Tris extender. Both sperm quality and MDA level decreased significantly (P < 0.05) from pre-freeze to post-thaw in both the extenders. CONCLUSION: The findings of the present study indicate that Ovixcell is a comparable alternative to Tris extender for the cryopreservation of crossbred ram semen.

Open pulled straw vitrification and slow freezing of sheep IVF embryos using different cryoprotectants.

The aim of the present study was to evaluate the post-thaw survival and hatching rates of sheep blastocysts using different cryoprotectants. In Experiment 1, Day 6 sheep embryos were cryopreserved by a slow freezing protocol using 10% ethylene glycol (EG), 10% dimethyl sulfoxide (DMSO) or a mixture of 5% EG and 5% DMSO. Hatching rates were higher in the 10% EG group than in the 10% DMSO or EG + DMSO groups (30% vs 18% and 20%, respectively). In Experiment 2, embryos were cryopreserved by open pulled straw (OPS) vitrification using either 33% EG, 33% DMSO or a mixture of 16.5% EG + 16.5% DMSO. Re-expansion and hatching rates in the EG + DMSO group (79.16% and 52.74%, respectively) were higher than those in the EG group (64.28% and 30.02%, respectively), whereas the outcomes for the DMSO group were the lowest (45.18% and 8.6%, respectively). In Experiment 3, embryos were cryopreserved by OPS vitrification using either 40% EG, 40% DMSO or a mixture of 20% EG + 20% DMSO. Re-expansion and hatching rates were highest in the EG group than in the EG + DMSO and DMSO groups (92.16% vs 76.30% and 55.84% re-expansion, respectively; and 65.78% vs 45.55% and 14.46% hatching, respectively). In conclusion, OPS vitrification was found to be more efficient for cryopreservation of in vitro-developed sheep embryos than traditional freezing.

Interaction of Al(2)O(3) nanoparticles with Escherichia coli and their cell envelope biomolecules.

AIMS: The aim of this study is to investigate the antibacterial activity of aluminium oxide nanoparticles (Al2 O3 NPs) against multidrug-resistant clinical isolates of Escherichia coli and their interaction with cell envelope biomolecules. METHODS AND RESULTS: Al2 O3 NPs were characterized by scanning electron microscope (SEM), high-resolution transmission electron microscope (HR-TEM) and X-ray diffraction (XRD) analyses. Antibacterial activity and interaction of Al2 O3 NPs with E. coli and its surface biomolecules were assessed by spectrophotometry, SEM, HR-TEM and attenuated total reflectance/Fourier transform infrared (ATR-FTIR). Of the 80 isolates tested, about 64 (80%) were found to be extended spectrum ß-lactamase (ESBL) positive and 16 (20%) were non-ESBL producers. Al2 O3 NPs at 1000 µg ml(-1) significantly inhibited the bacterial growth. SEM and HR-TEM analyses revealed the attachment of NPs to the surface of cell membrane and also their presence inside the cells due to formation of irregular-shaped pits and perforation on the surfaces of bacterial cells. The intracellular Al2 O3 NPs might have interacted with cellular biomolecules and caused adverse effects eventually triggering the cell death. ATR-FTIR studies suggested the interaction of lipopolysaccharide (LPS) and L-α-Phosphatidyl-ethanolamine (PE) with Al2 O3 NPs. Infrared (IR) spectral changes revealed that the LPS could bind to Al2 O3 NPs through hydrogen binding and ligand exchange. The Al2 O3 NPs-induced structural changes in phospholipids may lead to the loss of amphiphilic properties, destruction of the membrane and cell leaking. CONCLUSIONS: The penetration and accumulation of NPs inside the bacterial cell cause pit formation, perforation and disorganization and thus drastically disturb its proper function. The cell surface biomolecular changes revealed by ATR-FTIR spectra provide a better understanding of the cytotoxicity of Al2 O3 NPs. SIGNIFICANCE AND IMPACT OF THE STUDY: Al2 O3 NPs may serve as broad-spectrum bactericidal agents to control the emergent pathogens regardless of their drug-resistance mechanisms.

Comparison of slow freezing and vitrification on ovine immature oocytes.

BACKGROUND: Immature oocytes are more sensitive to cold injury than mature oocytes. OBJECTIVE: The study was to evaluate the post thaw normal oocytes, cleavage and blastocyst rates of ovine cumulus oocyte complexes (COC's) using different cryoprotectants by slow freezing and Open pulled straw (OPS) vitrification. METHODS: In five replicates, abattoir derived COC's were collected and distributed into three groups. In Experiment 1, COC's were cryopreserved by a slow freezing protocol using 10% concentration of ethylene glycol (EG), 10% dimethyl sulphoxide (DMSO) or 5% EG and 5% DMSO mixture. In Experiment 2 and 3 embryos were cryopreserved by OPS vitrification using either 33% or 40% (EG, DMSO or an equal mixture of EG and DMSO mixture. Normal oocytes post thaw were in vitro matured and parthenogenetically activated. RESULTS: Although, there was no difference in the number of post thaw normal oocytes between the groups, cleavage and blastocyst rates were higher in 10% slow freezing group than any of the vitrified groups. CONCLUSION: The study demonstrates better cryopreservation of ovine COC's by controlled slow freezing than OPS vitrification.

Biochemical alteration of hepatic functions by histamine H3-receptor agonist and antagonist in immunized rabbits.

AIM: The aim of our study was to investigate the functional roles of H3R agonist and antagonist in the development of hepatic functions impairment in immunized rabbits. METHODS: The study comprised of six groups containing 18 rabbits in each. Group-I (negative control) and group-II (positive control) received sterile distilled water intramuscularly while Group III-VI received histamine (100 µgkg-1, s.c.), R-[-]-α-methylhistamine (H3R-agonist, 10 µgkg-1, s.c.), iodophenpropit (H3R-antagonist, 1 µgkg-1, i.m.), and the combination of iodophenpropit (1 µgkg-1, i.m.) plus histamine (100 µgkg-1, s.c.), respectively, b.i.d. (12 hours [8 am and 8 pm]) for 10 days. Groups II-VI were immunized on day 3 with intravenous injection of sheep red blood cells (1×109 cells/ml). RESULTS: On each experimental day, the mean values of serum enzymes and bilirubin in group-I and group-II showed no changes while in groups III, IV, V, and VI, these enzymes and bilirubin levels showed significant changes (p<0.05), when compared with their values within the group. Profile of ALT and AST production revealed that ALT and AST levels moderately were changed due to degeneration of the liver. CONCLUSION: Our results suggest that R-[-]-α-methylhistamine showed moderate, and histamine and iodophenpropit showed mild degeneration of liver functions; while iodophenpropit plus histamine showed hepatic functions similar to control group. This study suggests that H3R antagonist in combination with histamine may be a non-toxic therapeutic target for histamine research (Fig. 7, Ref. 28). Text in PDF www.elis.sk.

Brolucizumab after failure of aflibercept with photodynamic therapy in polypoidal choroidal vasculopathy: A case report.

We describe one case of polypoidal choroidal vasculopathy with persistent subretinal fluid despite multiple treatment with intravitreal Bevacizumab, Ranibizumab and Aflibercept, as well as Aflibercept associated with photodynamic therapy. The patient reached complete resolution after intravitreal Brolucizumab injection, but experienced recurrence of subretinal fluid 12 weeks after discontinuation. Brolucizumab might be an option in treating subretinal fluid after failure of other anti-VEGF agents associated with photodynamic therapy.

Formulation development of a cream containing fennel extract: in vivo evaluation for anti-aging effects.

This study was aimed to formulate and evaluate anti-aging effects of a topical cream (w/o emulsion) containig extract of Fennel (Foeniculum vulgare) versus its base. Formulation containing 4% concentrated extract of Foeniculum vulgare was developed by entrapping in the inner aqueous phase of w/o emulsion and base contained no extract. Both the base and formulation were stored under different storage conditions to predict their stability. The formulation and base were evaluated for effect on skin moisture and transepidermal water loss (TEWL). The base showed insignificant while the formulation showed significant effects on skin moisture and TEWL. The parameter volume and surface evaluation of living skin (SELS) parameters SEr, SEsc, SEsm, SEw were also evaluated and showed a significant (p < or = 0.05) decline. The texture parameter energy showed a significant increase proving that the formulation possesses potential anti-aging effects.

In vivo study of histamine H4 receptor in immunomodulation.

OBJECTIVE: Recently accumulating evidence has highlighted the role of histamine in inflammation and immune reaction by histamine H4-receptor, however the role of histamine via H4-receptor in immunomodulation is still unclear. Therefore, the present study was designed to study the immunomodulatory role of histamine H4-receptor on antibody generation profile in rabbit. METHODS: The cohort study comprised of 108 rabbits in six groups. Each group consisted of 18 rabbits. Group I (negative control) remained non-immunized and received vehicle (sterile distilled water, 1 mlkg-1 × b.i.d., s.c. for 10 days (3 days prior to immunization until 7 days after immunization)). Group II (positive control) received vehicle (1 mlkg-1 × b.i.d., s.c. for 10 day), while group III-VI received histamine (100 µgkg-1 × b.i.d., s.c.), H4-agonist (clobenpropit dihydrobromide, 10 µgkg-1 × b.i.d., s.c.), and H4-antagonist (JNJ 7777120, 10 µgkg-1 × b.i.d., i.m.) and DMSO (control group for H4R-antagonist, 1 mlkg-1 × b.i.d., i.m.) respectively for 10 days. Group II-VI were immunized with intravenous injection of sheep red blood cells (SRBC) on day 3. Immunological parameters [immunoglobulins (Ig), immunoglobulin M (IgM), and immunoglobulin G (IgG)] assessed by the whole SRBC-ELISA method and direct hemagglutination assay. RESULTS: Histamine could influence a detectable antibody response to SRBC as early as day 7 postimmunization (post-I), which lasted until day 58 post-I, whereas H4-receptor by H4R-antagonist treatment showed a similar profile of antibody (Ig, IgM, and IgG) generation as the positive control group. On the other hand, H4R-agonist treatment showed immunostimulant activity as compared to other experimental groups. The results were found statistically significant (p<0.01). CONCLUSIONS: Histamine H4-receptor in biological system modulates immunological function and stimulates antibody production only by exogenously administered agonists not by endogenous histamine (Tab. 1, Fig. 3, Ref. 26).

Effect of age and season on semen quality parameters in Sahiwal bulls.

The objective of the study was to determine the effect of season, period, age, bull, and ejaculate on semen quality in Sahiwal bulls. Semen production records from 1996 to 2006 of 5,483 ejaculates from 46 Sahiwal bulls maintained at Artificial Breeding Complex, NDRI, Karnal, India were analyzed using least square analysis of variance by LSML software package. The overall least squares means of ejaculate volume (VOL), total volume per day (VOLD), mass activity (MA), initial motility (IM), sperm concentration per ml (SPC), and sperm concentration per ejaculate (SPCE) were 3.79 ± 0.02 ml, 5.81 ± 0.06 ml, 2.32 ± 0.01, 55.47 ± 0.001%, 766.69 ± 5.50 × 10(6)/ml and 3023.25 ± 30.15 × 10(6), respectively. All semen traits (VOL, VOLD, MA, IM and SPCE) were significantly (P < 0.01) affected by age groups, season and period, whereas season had significant effect on VOL at 5% level. During hot-humid season, highest value of VOL, VOLD, MA, IM, SPC, and SPCE were observed followed by summer and cold season. Highest value of VOL, VOLD, IM, and SPCE were observed during period-3 (2004-2006), whereas highest value of MA and SPC were observed during period-1 (1996-1999). However, lowest magnitude of MA, IM, SPC, and SPCE during period-2 (2000-2003) was observed. Ejaculate characteristics like VOL, VOLD, and SPCE increased with the increasing age of bull up to 5 years and then decreased. Significant (P < 0.01) bull to bull variation was found in VOL, VOLD, MA, IM, SPC, and SPCE traits. First ejaculate had significantly (P < 0.01) higher MA, IM, SPC, and SPCE. Hence, it could be concluded that during rainy season and period-1 and period-3 the quality of semen is quantitatively and qualitatively good. Better quality semen was obtained up to 5 years of age in Sahiwal bulls.

Insights into lipid-protein interactions from computer simulations.

Lipid-protein interactions play an important direct role in the function of many membrane proteins. We argue they are key players in membrane structure, modulate membrane proteins in more subtle ways than direct binding, and are important for understanding the mechanism of classes of hydrophobic drugs. By directly comparing membrane proteins from different families in the same, complex lipid mixture, we found a unique lipid environment for every protein. Extending this work, we identified both differences and similarities in the lipid environment of GPCRs, dependent on which family they belong to and in some cases their conformational state, with particular emphasis on the distribution of cholesterol. More recently, we have been studying modes of coupling between protein conformation and local membrane properties using model proteins. In more applied approaches, we have used similar methods to investigate specific hypotheses on interactions of lipid and lipid-like molecules with ion channels. We conclude this perspective with some considerations for future work, including a new more sophisticated coarse-grained force field (Martini 3), an interactive visual exploration framework, and opportunities to improve sampling.

Beta-lactams and beta-lactamase-inhibitors in current- or potential-clinical practice: a comprehensive update.

The use of successive generations of beta-lactams has selected successive generations of beta-lactamases including CTX-M ESBLs, AmpC beta-lactamases, and KPC carbapenamases in Enterobacteriaceae. Moreover, this cephalosporin resistance, along with rising resistance to fluoroquinolones, is now driving the use of carbapenems and unfortunately the carbapenem resistance has emerged markedly, especially in Acinetobacter spp. due to OXA- and metallo-carbapenemases. The industry responded to the challenge of rising resistance and recently developed some novel beta-lactams such as ceftobiprole, ceftaroline etc. and many beta-lactam compounds, including beta-lactamase-inhibitors, such as BMS-247243, S-3578, RWJ-54428, CS-023, SMP-601, NXL 104, BAL 30376, LK 157, and so on are under trials. This review provides the comprehensive accounts of the developments in penicillins, cephalosporins, carbapenems, and beta-lactamase-inhibitors, and the insight about medicinal chemistry, mechanism(s) of action and resistance, potential strategies to overcome resistance due to beta-lactamases, and also the recent advancements in the development of newer beta-lactam compounds; some of which are still under trials and yet to be classified. This review will fill the gap since previously published reviews and will serve as a comprehensive update on the current topic.

Anti-biofilm efficacy of silver nanoparticles against MRSA and MRSE isolated from wounds in a tertiary care hospital.

PURPOSE: Different approaches have been used for preventing biofilm-related infections in health care settings. Many of these methods have their own de-merits, which include chemical-based complications; emergent antibiotic resistant strains, etc. The formation of biofilm is the hallmark characteristic of Staphylococcus aureus and S. epidermidis infection, which consists of multiple layers of bacteria encased within an exopolysachharide glycocalyx. Nanotechnology may provide the answer to penetrate such biofilms and reduce biofilm formation. Therefore, the aim of present study was to demonstrate the biofilm formation by methicillin resistance S. aureus (MRSA) and methicillin resistance S. epidermidis (MRSE) isolated from wounds by direct visualisation applying tissue culture plate, tube and Congo Red Agar methods. MATERIALS AND METHODS: The anti-biofilm activity of AgNPs was investigated by Congo Red, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) techniques. RESULTS: The minimum inhibitory concentration (MIC) was found to be in the range of 11.25-45 µg/ml. The AgNPs coated surfaces effectively restricted biofilm formation of the tested bacteria. Double fluorescent staining (propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A (Con A-FITC) staining to detect the exopolysachharides matrix) technique using CLSM provides the visual evidence that AgNPs arrested the bacterial growth and prevent the glycocalyx formation. In our study, we could demonstrate the complete anti-biofilm activity AgNPs at a concentration as low as 50 µg/ml. CONCLUSIONS: Our findings suggested that AgNPs can be exploited towards the development of potential anti-bacterial coatings for various biomedical and environmental applications. In the near future, the AgNPs may play major role in the coating of medical devices and treatment of infections caused due to highly antibiotic resistant biofilm.

Molecular characterization of RNA binding motif protein 3 (RBM3) gene from Pashmina goat.

Pashmina goat inhabits the high altitude cold arid desert of Ladakh, India. This goat is known for its finest and costliest under fiber. Though the under fiber may be a part of its complex thermoregulation mechanism, the genetics of its adaptability under cold conditions is not known. As an attempt to understand its adaptive genetics, and the role of RNA-binding proteins at the cellular response, this study was conducted to characterize the RBM3 gene in Pashmina goat and its expression during hypothermia. The ORF of Pashmina RBM3 gene was 273 bp. Phylogenetic analysis revealed that Pashmina RBM3 is closely related to Bos taurus RBM3. Pashmina RBM3 was characterized by comparative modeling studies. The final 3-D model contained two α-helices and four ß-sheets. qRT-PCR data showed that Pashmina RBM3 gene expression was significantly higher (P < 0.05) at moderate (30 °C) hypothermic stress conditions as compared with deep (15 °C) hypothermia.

Rapid diagnosis of typhoid fever by co-agglutination in an Indian hospital.

Detection of Salmonella typhi infection by a co-agglutination assay for specific O, H and Vi antigens and by blood culture were compared for 110 patients with suspected typhoid fever. Blood cultures were positive for S. typhi in 25.5% of patients. Co-agglutination tests with patients' serum and with blood culture supernates gave positive results in 70.9% and 67.3% of cases respectively. S. typhi antigens Hd and O9 were detected in patients' serum by co-agglutination in 96.4% of blood culture-positive, and 62.2% of blood culture-negative patients. Co-agglutination results were uniformly negative with serum samples from a control group of 50 healthy individuals, 20 patients with febrile non-typhoid infectious disease and 20 patients with non-infectious febrile disease. Of the 25 patients with suspected typhoid fever who had not received prior antibiotic treatment, 88% yielded positive blood cultures and 96% gave positive results in serum co-agglutination tests. By contrast, of the 85 patients who had received prior antibiotics, only 7% yielded positive blood cultures, but 63.5% gave positive results in serum co-agglutination tests. Co-agglutination tests with serum offer a simple, rapid, sensitive, specific and economical method for the early diagnosis of typhoid fever.

Biochemical studies on mouse liver following Plasmodium berghei infection.

The lipid composition of mouse liver following infection with P. berghei was investigated. The liver lipid contents of infected animals were greatly increased mainly due to the accumulation of triacylglycerides. There was enhanced lipid concentration (85.29%). Significantly (23.7%) depleted liver cholesterol was also found in the mice. Similarly, phospholipid contents of liver were also decreased by 19.90 per cent. The liver from P. berghei infected mouse produced more lipid peroxide, as compared to control animals (314%). Significant depletion was also observed in carbohydrate, glycogen and glucose (79.1, 86.26 and 78.6% respectively) contents of liver at high parasitaemia. The lower contents of nucleic acid in the infected hosts observed in the study may be partly due to the absorption of nucleic acids by the parasites from the host cells.

Natural gamma-emiting radionuclides in Pakistani Portland cement.

Studies of the natural gamma-emitting radionuclides in Portland cement manufactured in the North West Frontier Province (NWFP) of Pakistan and the various raw materials which compose the product have been carried out using gamma-spectrometric techniques. For data acquisition a high-purity germanium detector (HPGe) was used. The range of the total specific activity (minimum and maximum values) due to all the three radionuclides (40K, 226Ra and 232Th) were found to be 187.8+/-63.5-573.2+/-73.1 Bq kg(-1) (Portland cement); 54.5+/-16.1-183.9+/-31.4 Bq kg(-1) (limestone); 87.1+/-30.7-297.1+/-64.4 Bq kg(-1) (gypsum); 696.4+/-79.1-1043.9+/-85.0 Bq kg(-1) (slate); and 490.9+/-54.5-570.2+/-59.8 Bq kg(-1) (latrite). The average specific activities due to 40K in Portland cement and all the raw materials were found to be higher when compared with 226Ra and 232Th. Such materials do not pose any excess radiological health problem.

Norm and associated radiation hazards in bricks fabricated in various localities of the North-West Frontier Province (Pakistan).

The activity concentrations of natural gamma-emitting radionuclides and associated radiation hazards due to 40K, 226Ra and 232Th have been measured in baked brick samples, collected from six highly populated areas of the North-West Frontier Province (NWFP) of Pakistan. For the detection, analysis and data acquisition, a high purity germanium (HPGe) detector coupled with a high resolution multichannel analyser (MCA) was used. The range of the average values of the activity concentrations due to 40K, 226Ra and 232Th were found to be 680.3 +/- 22.2-784.4 +/- 30.7 Bq kg(-1), 36.9 +/- 3.5-51.9 +/- 3.3 Bq kg(-1) and 52.5 +/- 3.6-67.6 +/- 3.1 Bq kg(-1), respectively. Radium equivalent (Ra(eq)) activities and various hazard indices were also calculated to assess the radiation hazards. All the brick samples showed Ra, activities within the limit (370 Bq kg(-1)) set by the Organization for Economic Cooperation and Development (OECD) countries. The results of different criterion formulae also complemented each other in this study. The derived data have been compared with the reported values for other countries of the world.

Radiological significance of building bricks in Pakistan.

Baked clay bricks samples were collected from both urban and rural areas of the North West Frontier Province of Pakistan and natural radioactivity due to 40K, 226Ra and 232Th has been measured in these samples. The specific absorbed dose rates due to natural radioactivity emanating from the walls, floors and ceilings of the typical model rooms made of these building bricks have been calculated applying the mesh size adaptive volume integral methodology. The occupancy factor, representing the weighted average of all the population in the country, was taken to be 0.3 in this study. The values of estimated annual effective dose rates for whole body were found to be 0.38 and 0.35 mSv.y(-1) for the two types of model rooms. These values are smaller than those predicted by UNSCEAR for normal background areas. Thus the building bricks analysed in this study do not pose any significant health problem to the population.