Ilmenite-derived titanic acid species: exploring their outstanding light-independent antibacterial activity.

The emergence of resistance in detrimental pathogenic bacteria towards well-recognized antibiotics has greatly impacted global medicine, consequently exploring potent antibacterial compounds is becoming a potential area of research. Although photocatalytic metal oxides have been extensively explored in this regard, their applicability is diminished due to the requirement of photon energy. Therefore, in our study, we explored the light-independent antibacterial effect of two unexplored titanium species, known as metatitanic acid (MTA) and potassium titanate, against Staphylococcus aureus, Escherichia coli, and Pseudomonas spp. using the disk diffusion method in Luria-Bertani agar medium, where the well-known antibiotic, gentamicin, was used as the positive control. These two titanium compounds were readily synthesized through a novel process which was originally developed for the extraction of TiO2 from ilmenite. The synthesized MTA was characterized using FT-IR, Raman spectroscopy, XRD, TGA, UV-visible spectroscopy, and SEM. According to our findings, both MTA and potassium titanate exhibited superior light-independent antibacterial properties, where for some concentrations, the effect was even greater than gentamicin. However, nano-TiO2 totally failed as an antibacterial compound against the tested three strains under dark conditions.

Possible cerebroprotective effect of citronellal: molecular docking, MD simulation and in vivo investigations.

This study focused on molecular docking, dynamic simulation, and in vivo approaches to examine the molecular interactions between citronellal (CT) and neurotoxic proteins. In silico studies of CT were performed using proteins involved in the pathophysiology of stroke, such as interleukin-6 (IL-6), interleukin-12 (IL-12), TNF-α, and nitric oxide synthase (NOS), to determine the binding affinity based on their interactions. The docking results of CT revealed that, among the targets, NOS had a better binding energy of -6.4 Kcal/mol. NOS showed good hydrophobic interactions: TYR A, 347; VAL A, 352; PRO A, 350; TYR A, 373 amino acids. Interactions with IL-6, TNF-α, and IL-12 resulted in lower binding affinities of -3.7, -3.9 and -3.1 Kcal/mol. Based on molecular dynamics simulations of 100 ns, the binding affinity of CT (-66.782 ± 7.309 kJ/mol) was well complemented, and NOS stability at the docked site was confirmed. In in vivo studies, cerebral stroke was induced by occlusion of the bilateral common carotid arteries for 30 min and reperfusion for 4 h. CT treatment protected the brain by decreasing cerebral infarction size, increasing GSH(p < 0.001***), decreasing MPO (p < 0.001***), MDA (p < 0.001***), NO production (p < 0.01**), and AChE (p < 0.001***) compared to stroke rats. Histopathological examination revealed that CT treatment reduced the severity of cerebral damage. The investigation concluded that CT strongly binds to NOS, as observed in molecular docking and dynamic simulation studies, which are involved in nitric oxide production, leading to cerebral damage, and CT treatment reduces NO production and oxidative stress parameters, and increases antioxidants via inhibition of NOS function.Communicated by Ramaswamy H. Sarma.

Integrating the Carboxylate Platform into a Red Seaweed Biorefinery.

Macroalgae are an important source of food, fertilizer, hydrocolloids, and healthful bioactive components. Macroalgae are also being considered sources of biofuels, which require minimal demands for arable land, fresh water, or fertilizers. In this study, we explored the possibility of developing a red seaweed biorefinery process to extract carrageenan while producing chemical or biofuel co-products derived from the carrageenan extraction wastes. A common approach to processing organic wastes is to generate biogas; however, in this study, we targeted a potentially higher value option by applying acidogenic digestion to convert extraction wastes to carboxylic acids and derived compounds. Using an open culture of microorganisms, wastes from a carrageenan extraction plant were converted to mixed carboxylic acids, which were then neutralized and thermally decomposed to a variety of ketones. Batch digestions of the wastes were carried out at temperatures of 35 °C and 55 °C. Either calcium carbonate or ammonium bicarbonate was used as buffer. A solid-liquid counter-current percolation fermentation was operated in four stages at 35 °C. Digestion produced carboxylic acids ranging in chain length from one to seven carbons. The mesophilic temperature gave higher carboxylic acid yield and longer chain acids, with the highest acid titer reaching 18 g L-1. Thermal decomposition of carboxylate salts produced a mixture of ketones which contained acetone, 3-pentanone, 2-hexanone, 2-heptanone, 3-heptanone, and 4-octanone as major products. These ketones could be sold as chemicals or hydrogenated to form corresponding chain length secondary alcohols which deliver higher energy density than ethanol.

The genotypes and virulence attributes of C. albicans isolates from oral leukoplakia.

BACKGROUND: There is a debate as to whether some types of oral leucoplakias (OL) are caused by Candida species, and whether they contribute to the malignant transformation, associated with a minority of such lesions. As no detailed population analysis of yeast isolates from OL is available, we evaluated the virulence attributes, and genotypes of 35 C. albicans from OL, and compared their genotypes with 18 oral isolates from healthy individuals. MATERIAL AND METHODS: The virulence traits evaluated were esterase, phospholipase, proteinase, haemolysin and coagulase production, and phenotypic switching activity, and yeast adherence and biofilm formation. DNA from OL and control yeasts were evaluated for A, B or C genotype status. RESULTS: Phospholipase, proteinase, and coagulase activity and biofilm formation was observed in 80%, 66%, 97 % and 77 % of the isolates, respectively. Phenotypic switching was detected in 8.6%, while heamolytic, and esterase activity and adherence were noted in all isolates. CONCLUSIONS: The genotype A was predominant amongst both the OL and control groups. Due to the small sample size of our study a larger investigation to define the role of candidal virulent attributes in the pathogenicity of OL is warranted, and the current data should serve as a basis until then.

No Evidence for Trade-Offs Between Lifespan, Fecundity, and Basal Metabolic Rate Mediated by Liver Fatty Acid Composition in Birds.

The fatty acid composition of biological membranes has been hypothesised to be a key molecular adaptation associated with the evolution of metabolic rates, ageing, and life span - the basis of the membrane pacemaker hypothesis (MPH). MPH proposes that highly unsaturated membranes enhance cellular metabolic processes while being more prone to oxidative damage, thereby increasing the rates of metabolism and ageing. MPH could, therefore, provide a mechanistic explanation for trade-offs between longevity, fecundity, and metabolic rates, predicting that short-lived species with fast metabolic rates and higher fecundity would have greater levels of membrane unsaturation. However, previous comparative studies testing MPH provide mixed evidence regarding the direction of covariation between fatty acid unsaturation and life span or metabolic rate. Moreover, some empirical studies suggest that an n-3/n-6 PUFA ratio or the fatty acid chain length, rather than the overall unsaturation, could be the key traits coevolving with life span. In this study, we tested the coevolution of liver fatty acid composition with maximum life span, annual fecundity, and basal metabolic rate (BMR), using a recently published data set comprising liver fatty acid composition of 106 avian species. While statistically controlling for the confounding effects of body mass and phylogeny, we found no support for long life span evolving with low fatty acid unsaturation and only very weak support for fatty acid unsaturation acting as a pacemaker of BMR. Moreover, our analysis provided no evidence for the previously reported links between life span and n-3 PUFA/total PUFA or MUFA proportion. Our results rather suggest that long life span evolves with long-chain fatty acids irrespective of their degree of unsaturation as life span was positively associated with at least one long-chain fatty acid of each type (i.e., SFA, MUFA, n-6 PUFA, and n-3 PUFA). Importantly, maximum life span, annual fecundity, and BMR were associated with different fatty acids or fatty acid indices, indicating that longevity, fecundity, and BMR coevolve with different aspects of fatty acid composition. Therefore, in addition to posing significant challenges to MPH, our results imply that fatty acid composition does not pose an evolutionary constraint underpinning life-history trade-offs at the molecular level.

Identification and characterization of potential impurities of valsartan, AT1 receptor antagonist.

Five impurities (related substances) were detected during the impurity profile study of an antihypertensive drug substance, valsartan. A simple gradient high performance liquid chromatographic method (HPLC) and liquid chromatography-mass spectrometry (LC-MS) were used for the detection. Based on the spectral data (IR, NMR and MS) followed by synthesis, these impurities were characterized as (S)-N-(1-carboxy-2-methylprop-1-yl)-N-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]amine (impurity I); (S)-N-(1-carboxy-2-methylprop-1-yl)-N-(5-phenylthio)pentanoyl-N-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]amine (impurity II); (S)-N-(1-carboxy-2-methylprop-1-yl)-N-(5-phenyl)pentanoyl-N-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]amine (impurity III); (S)-N-(1-carboxy-2-methylprop-1-yl)-N-4-pentenoyl-N-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]amine (impurity IV); (S)-N-(1-carboxy-2-methylprop-1-yl)-N-(5-hydroxy)pentanoyl-N-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]amine (impurity V).

Use of the QuantiFERON-TB Gold In-Tube test to monitor treatment efficacy in active pulmonary tuberculosis.

SETTING: Cohort study at a tertiary care hospital. OBJECTIVE: To assess the potential use of QuantiFERON-TB Gold In-Tube (QFT-G) in monitoring clinical response to anti-tuberculosis treatment. DESIGN: We conducted a cohort study of 76 active pulmonary tuberculosis patients with serial testing by QFT-G at baseline and after 2 and 6 months of treatment. At these time points, we compared the performance of QFT-G with sputum culture status of the study subjects. RESULTS: Compared to baseline, 59 (77.6%) cases showed a decline whereas 17 (22.4%) showed persistent or stronger interferon-gamma (IFN-gamma) responses at 2 months. Using robust statistical methods, we observed that QFT-G assessment at 2 months independently and significantly predicted the likelihood of remaining sputum culture-positive at the end of the intensive phase of anti-tuberculosis treatment. A higher IFN-gamma concentration by 1 international unit (IU)/ml corresponded to a 45% (95%CI 8-97) higher likelihood of failing to convert to a negative culture, whereas a rising or persistent IFN-gamma response was associated with a 17.3 (P = 0.007) times higher likelihood of remaining culture-positive at 2 months. CONCLUSIONS: Our results suggest that QFT-G can potentially be used as a tool to monitor the efficacy of anti-tuberculosis treatment.

Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice.

A 10 microm spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo-3, fluo-4 or fluo-5F, to estimate dark, resting free Ca(2+) concentration ([Ca(2+)](i)) and changes in [Ca(2+)](i) upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild-type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin alpha-subunit knock-out (Tralpha-/-) animals showed no light-dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (tau~1-4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre-exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in [Ca(2+)](i). A similar rapid increase in fluorescence was also seen in the rods of wild-type mice just preceding the fall in fluorescence produced by the light-dependent decrease in [Ca(2+)](i). Dissociation constants were measured in vitro for fluo-3, fluo-4 and fluo-5F with and without 1 mM Mg(2+) from 20 to 37 degrees C. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3-4 over this range. Values at 37 degrees C were used to estimate absolute levels of rod [Ca(2+)](i). All three dyes gave similar values for [Ca(2+)](i) in wild-type rods of 250 +/- 20 nM in darkness and 23 +/- 2 nM after exposure to saturating light. There was no significant difference in dark [Ca(2+)](i) between wild-type and Tralpha-/- animals.

Both Rb and E7 are regulated by the ubiquitin proteasome pathway in HPV-containing cervical tumor cells.

High-risk human papillomaviruses (HPVs) are etiologically linked to human cervical and oral cancers. The E6 and E7 oncoproteins encoded by HPV target host cell tumor suppressor proteins. E6 induces proteolysis of p53 through the ubiquitin-proteasome pathway. Recent studies showed that overexpression of E7 caused proteolytic degradation of the tumor suppressor Rb. However, unlike p53, Rb is not regulated by proteolysis in normal cells. In addition, it was unclear whether in its natural context E7 regulates Rb through the ubiquitin-proteasome pathway. Therefore, we sought to determine whether Rb is regulated by the ubiquitin-proteasome pathway in HPV-containing tumor cells. We carried out a detailed analysis in Caski cells, that are derived from HPV-containing cervical cancer tissues. Studies with various protease inhibitors revealed that Rb is regulated specifically by the ubiquitin-proteasome pathway in HPV-containing cervical tumor cells. Several inhibitors of the 26S proteasome significantly increased the level of Rb in the Caski cells. Rb controls cell growth by forming complexes with the E2F-family transcription factors. Surprisingly, in spite of a significant accumulation of the hypophosphorylated form of Rb, no Rb/E2F complex was detectable in the proteasome inhibitor treated cells. Further analysis revealed that there was an increased accumulation of the E7 oncoprotein. We showed that the proteasome inhibitors simultaneously blocked the proteolysis of E7 and Rb, suggesting that E7 is also regulated by the ubiquitin-dependent proteolysis in cervical cancer cells. Taken together, this study suggests that targeted inhibition of Rb proteolysis will be required for restoring Rb function in HPV-containing cervical cancer cells.

Mapping of hiv-1 Gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system.

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50-250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions. The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.

Simplified gene-fragment phage display system for epitope mapping.

We describe a simple and efficient system for epitope mapping by cloning random gene fragments into a specially designed gIIIp-based phage display vector. DNA encoding the antigen of interest is PCR-amplified and partially digested with DNaseI to generate 50-150-bp-long fragments, which are polished with T4 DNA polymerase and dephosphorylated. These fragments are cloned at the 5' end of the gIII after linearizing the vector with SmaI/SrfI, and the ligation is carried out in the presence of restriction enzyme SrfI. The restriction enzyme in the ligation reaction recuts the self-ligated vector but not the recombinants, since ligation with foreign fragments destroys the enzyme recognition site. Dephosphorylation of inserts prevents their chimerization and ensures ligation of single insert per vector molecule. Thus, using the above strategy, which prevents self-ligation of both the insert and the vector, the overall cloning efficiency and, thereby the library size, is improved more than 10-fold compared to the standard blunt-end, ligation-based methods for making similar libraries. The library is further enriched by a single-step infection of E. coli by phages obtained from primary transformants. This step eliminates all the phages that carry insert that are not in-frame with gIIIp and therefore do not display gIIIp. We have shown the utility of the above system in constructing a glutathione-S-transferase (GST) gene-fragment library in phages and identifying the epitope recognized by a monoclonal antibody against GST.

Cloning and characterization of RNF6, a novel RING finger gene mapping to 13q12.

Myeloproliferative disorders frequently show deletions or rearrangements of the long arm of chromosome 13. We report here the cloning of RNF6, a new gene that maps close to the chromosome 13 breakpoint in a case of myelofibrosis with a t(4;13)(q26;q12). RNF6 is predicted to encode a 685-amino-acid protein with a coiled-coil domain and a RING-H2 finger at the amino and carboxy terminis, respectively. In addition, we have identified a novel motif, Lys-X-X-Leu/Ile-X-X-Leu/Ile (KIL motif), that is located shortly upstream of a subset of RING-H2 proteins, including RNF6. Drosophila g1, rat Neurodap1, and mouse Praja1. FISH and physical mapping indicated that RNF6 is located at 13q12.2 close to marker D13S1121, and it is oriented from telomere to centromere. RNF6 is not disrupted by the t(4;13).

Light-dependent changes in outer segment free-Ca2+ concentration in salamander cone photoreceptors.

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 +/- 2.4 and 640 +/- 55 ms (mean +/- SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 +/- 30 and 1,400 +/- 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 +/- 37 nM in the dark-adapted outer segment and 5.5 +/- 2.4 nM after saturating illumination (mean +/- SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

Bleached pigment produces a maintained decrease in outer segment Ca2+ in salamander rods.

A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+i estimates of 670 +/- 250 nM in a dark-adapted rod and 30 +/- 10 nM during response saturation after exposure to bright light (mean +/- SD). The resting level of Ca2+i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 +/- 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

Versatile vectors for direct cloning and ligation-independent cloning of PCR-amplified fragments for surface display on filamentous bacteriophages.

We have constructed phagemid and phage-based vectors which can be used for both direct (T/A) and ligation-independent cloning (LIC) of PCR products for surface display of encoded peptides/proteins fused with the gIII protein of the filamentous bacteriophages M13 and fd-tet. The vectors harbour a DNA cassette consisting of the lacZ alpha fragment inserted between the +2 and +3 codons of gIIIp. The lacZ alpha fragment is flanked by several restriction enzyme recognition sites which can be used for conventional blunt- and cohesive-end cloning in addition to T/A cloning and LIC. The cloning strategies lead to the loss of the lacZ alpha fragment facilitating the selection of the recombinants in XGal plates. The efficiency of direct (T/A) cloning and LIC for surface display in both vectors was evaluated using PCR-amplified fragments encoding a variety of different proteins which included the Fc-binding domain of protein A, the ADP-ribosylation domain of Pseudomonas exotoxin A and a single-chain antibody fragment. The cloning efficiency obtained was 75-85% using the two strategies as monitored by restriction enzyme analysis of the recombinant white colonies on XGal plates. The expression of encoded proteins in recombinants, which were displayed as gIIIp fusions, was found to be 10% in case of T/A cloning but more than 90% in case of LIC.

Identification of new HLA class I region genes by sample sequencing.

Although many human major histocompatibility genes have been identified, relatively few have been localized to the class I region. We searched for new class I region genes by sample sequencing, a process in which short stretches of random genomic sequence are generated from cosmids and then compared with sequences deposited in nucleotide databases. Four class I region cosmids were isolated for sample sequencing by screening a chromosome 6 specific cosmid library with probes derived from specific class I region genes or with overlapping class I region yeast artificial chromosomes. Cosmids were sonnicated to produce fragments of 0.5 - 1 kilobases, subcloned, and sequenced using an automated sequencer. Sequences were then compared with nucleotide sequences deposited in the GenBank databases using the BLASTN algorithm. A number of potential new class I region genes were identified, including a cDNA with similarity to the tre oncogene, the trans-activating factor SC1 (TCF19), and a member of the interferon inducible 1 - 8 gene family. These observations suggest that sample sequencing is an efficient method for identifying new class I region genes, which can be applied to other regions of the genome and to other species, and support previous observations that the class I region contains a variety of genes other than those encoding HLA antigens.

Genetic mapping of the human homologue (T) of mouse T(Brachyury) and a search for allele association between human T and spina bifida.

We describe a genetic analysis of the human homologue (T) of the mouse T (Brachyury) gene; human T was recently cloned in our laboratory. The protein product of the T gene is a transcription factor crucial in vertebrates for the formation of normal mesoderm. T mutant Brachyury mice die in midgestation with severe defects in posterior mesodermal tissues; heterozygous mice are viable but have posterior axial malformations. In addition to its importance in development, T has intrigued geneticists because of its association with the mouse t-haplotype; this haplotype is a variant form of the t-complex and is characterized by transmission ratio distortion, male sterility and recombination suppression. We have identified a common polymorphism of human T by single strand conformation polymorphism (SSCP) and used this in mapping studies and to re-investigate the idea that human T is involved in susceptibility to the multifactorial, neural tube defect, spina bifida. Our mapping data show that human T maps to 6q27 and lies between two other genes of the t-complex, TCP1 and TCP10. These data add to the evidence that in man the genes of the t-complex are split into two main locations on the short and long arms of chromosome 6. We have used an allele association test which is independent of mode of inheritance and penetrance to analyse data from the spina bifida families. Using this test we find evidence for a significant (p = 0.02) association between transmission of the TIVS7-2 allele of the human T gene and spina bifida.