Air-kerma evaluation at the maze entrance of HDR brachytherapy facilities.

In the absence of procedures for evaluating the design of brachytherapy (BT) facilities for radiation protection purposes, the methodology used for external beam radiotherapy facilities is often adapted. The purpose of this study is to adapt the NCRP 151 methodology for estimating the air-kerma rate at the door in BT facilities. Such methodology was checked against Monte Carlo (MC) techniques using the code Geant4. Five different facility designs were studied for (192)Ir and (60)Co HDR applications to account for several different bunker layouts.For the estimation of the lead thickness needed at the door, the use of transmission data for the real spectra at the door instead of the ones emitted by (192)Ir and (60)Co will reduce the lead thickness by a factor of five for (192)Ir and ten for (60)Co. This will significantly lighten the door and hence simplify construction and operating requirements for all bunkers.The adaptation proposed in this study to estimate the air-kerma rate at the door depends on the complexity of the maze: it provides good results for bunkers with a maze (i.e. similar to those used for linacs for which the NCRP 151 methodology was developed) but fails for less conventional designs. For those facilities, a specific Monte Carlo study is in order for reasons of safety and cost-effectiveness.

Sceletium tortuosum: A review on its phytochemistry, pharmacokinetics, biological, pre-clinical and clinical activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Sceletium tortuosum (L.) N.E.Br., the most sought after and widely researched species in the genus Sceletium is a succulent forb endemic to South Africa. Traditionally, this medicinal plant is mainly masticated or smoked and used for the relief of toothache, abdominal pain, as a mood-elevator, analgesic, hypnotic, anxiolytic, thirst and hunger suppressant, and for its intoxicating/euphoric effects. Sceletium tortuosum is currently of widespread scientific interest due to its clinical potential in treating anxiety and depression, relieving stress in healthy individuals, and enhancing cognitive functions. These pharmacological actions are attributed to its phytochemical constituents referred to as mesembrine-type alkaloids. AIM OF THE REVIEW: The aim of this review was to comprehensively summarize and critically evaluate recent research advances on the phytochemistry, pharmacokinetics, biological, pre-clinical and clinical activities of the medicinal plant S. tortuosum. Additionally, current ongoing research and future perspectives are also discussed. METHODS: All relevant scientific articles, books, MSc and Ph.D. dissertations on botany, behavioral pharmacology, traditional uses, and phytochemistry of S. tortuosum were retrieved from different databases (including Science Direct, PubMed, Google Scholar, Scopus and Web of Science). For pharmacokinetics and pharmacological effects of S. tortuosum, the focus fell on relevant publications published between 2009 and 2021. RESULTS: Twenty-five alkaloids belonging to four structural classes viz: mesembrine, Sceletium A4, joubertiamine, and tortuosamine, have been identified from S. tortuosum, of which the mesembrine class is predominant. The crude extracts and commercially available standardized extracts of S. tortuosum have displayed a wide spectrum of biological activities (e.g. antimalarial, anti-oxidant, neuromodulatory, immunomodulatory, anti-HIV, neuroprotection) in in vitro or in vivo studies. While the plant has been studied in clinical populations, this has only been in healthy subjects, so that further study in pathological states remains to be done. Nevertheless, the aforementioned studies have demonstrated that S. tortuosum has potential for enhancing cognitive function and managing anxiety and depression. CONCLUSION: As an important South African medicinal plant, S. tortuosum has garnered many research advances on its phytochemistry and biological activities over the last decade. These scientific studies have shown that S. tortuosum has various bioactivities. The findings have further established the link between the phytochemistry and pharmacological application, and support the traditional use of S. tortuosum in the indigenous medicine of South Africa.

Resolving voltage-dependent structural changes of a membrane photoreceptor by surface-enhanced IR difference spectroscopy.

Membrane proteins are molecular machines that transport ions, solutes, or information across the cell membrane. Electrophysiological techniques have unraveled many functional aspects of ion channels but suffer from the lack of structural sensitivity. Here, we present spectroelectrochemical data on vibrational changes of membrane proteins derived from a single monolayer. For the seven-helical transmembrane protein sensory rhodopsin II, structural changes of the protein backbone and the retinal cofactor as well as single ion transfer events are resolved by surface-enhanced IR difference absorption spectroscopy (SEIDAS). Angular changes of bonds versus the membrane normal have been determined because SEIDAS monitors only those vibrations whose dipole moment are oriented perpendicular to the solid surface. The application of negative membrane potentials (DeltaV = -0.3 V) leads to the selective halt of the light-induced proton transfer at the stage of D75, the counter ion of the retinal Schiff base. It is inferred that the voltage raises the energy barrier of this particular proton-transfer reaction, rendering the energy deposited in the retinal by light excitation insufficient for charge transfer to occur. The other structural rearrangements that accompany light-induced activity of the membrane protein, are essentially unaffected by the transmembrane electric field. Our results demonstrate that SEIDAS is a generic approach to study processes that depend on the membrane potential, like those in voltage-gated ion channels and transporters, to elucidate the mechanism of ion transfer with unprecedented spatial sensitivity and temporal resolution.

Sceletium tortuosum: A review on its phytochemistry, pharmacokinetics, biological and clinical activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Sceletium tortuosum (L.) N.E.Br, the most sought after and widely researched species in the genus Sceletium is a succulent forb endemic to South Africa. Traditionally, this medicinal plant is mainly masticated or smoked and used for the relief of toothache, abdominal pain, and as a mood-elevator, analgesic, hypnotic, anxiolytic, thirst and hunger suppressant, and for its intoxicating/euphoric effects. Sceletium tortuosum is currently of widespread scientific interest due to its clinical potential in treating anxiety and depression, relieving stress in healthy individuals, and enhancing cognitive functions. These pharmacological actions are attributed to its phytochemical constituents referred to as mesembrine-type alkaloids. AIM OF THE REVIEW: The aim of this review was to comprehensively summarize and critically evaluate recent research advances on the phytochemistry, pharmacokinetics, biological and clinical activities of the medicinal plant S. tortuosum. Additionally, current ongoing research and future perspectives are also discussed. METHODS: All relevant scientific articles, books, MSc and Ph.D. dissertations on botany, behavioral pharmacology, traditional uses, and phytochemistry of S. tortuosum were retrieved from different databases (including Science Direct, PubMed, Google Scholar, Scopus and Web of Science). For pharmacokinetics and pharmacological effects of S. tortuosum, the focus fell on relevant publications published between 2009 and 2021. RESULTS: Twenty-five alkaloids belonging to four structural classes viz: mesembrine, Sceletium A4, joubertiamine, and tortuosamine, have been identified from S. tortuosum, of which the mesembrine class is predominant. The crude extracts and commercially available standardized extracts of S. tortuosum have displayed a wide spectrum of biological activities (e.g. antimalarial, anti-oxidant, immunomodulatory, anti-HIV, neuroprotection, enhancement of cognitive function) in in vitro or in vivo studies. This plant has not yet been studied in a clinical population, but has potential for enhancing cognitive function, and managing anxiety and depression. CONCLUSION: As an important South African medicinal plant, S. tortuosum has garnered many research advances on its phytochemistry and biological activities over the last decade. These scientific studies have shown that S. tortuosum has various bioactivities. The findings have further established the link between the phytochemistry and pharmacological application, and support the traditional use of S. tortuosum in the indigenous medicine of South Africa.

Fire and herbivory drive fungal and bacterial communities through distinct above- and belowground mechanisms.

Fire and herbivory are important natural disturbances in grassy biomes. Both drivers are likely to influence belowground microbial communities but no studies have unravelled the long-term impact of both fire and herbivory on bacterial and fungal communities. We hypothesized that soil bacterial communities change through disturbance-induced shifts in soil properties (e.g. pH, nutrients) while soil fungal communities change through vegetation modification (biomass and species composition). To test these ideas, we characterised soil physico-chemical properties (pH, acidity, C, N, P and exchangeable cations content, texture, bulk density, moisture), plant species richness and biomass, microbial biomass and bacterial and fungal community composition and diversity (using 16S and ITS rRNA amplicon sequencing, respectively) in six long-term (18 to 70 years) ecological research sites in South African savanna and grassland ecosystems. We found that fire and herbivory regimes profoundly modified soil physico-chemical properties, plant species richness and standing biomass. In all sites, an increase in woody biomass (ranging from 12 to 50%) was observed when natural disturbances were excluded. The intensity and direction of changes in soil properties were highly dependent on the topo-pedo-climatic context. Overall, fire and herbivory shaped bacterial and fungal communities through distinct driving forces: edaphic properties (including Mg, pH, Ca) for bacteria, and vegetation (herbaceous biomass and woody cover) for fungi. Fire and herbivory explained on average 7.5 and 9.8% of the fungal community variability, respectively, compared to 6.0 and 5.6% for bacteria. The relatively small changes in microbial communities due to natural disturbance is in stark contrast to dramatic vegetation and edaphic changes and suggests that soil microbial communities, having evolved with disturbance, are resistant to change. This represents both a buffer to short-term anthropogenic-induced changes and a restoration challenge in the face of long-term changes.

[Lambliasis as differential diagnosis of MARSH type 3b]. / Lamblieninfektion als Differenzialdiagnose der Zöliakie in der Duodenalbiopsie.

Giardia lamblia is the most common human parasite with a worldwide distribution and fecal-oral way of transmission. Diagnostic procedures include stool examination and gastroduodenoscopy with biopsy or secret aspiration. In most cases histology reveals a dense accumulation of the parasites on the surface of the duodenal mucosa with no or only slight inflammation. In rare cases, a dense inflammatory infiltrate with severe mucosal atrophy and increased count of intraepithelial lymphocytes may be seen. If in such cases the amount of parasites is low, the histological picture may mimic celiac disease. The two presented cases demonstrate the close morphological relationship and show the importance of considering giardiasis in the differential diagnosis in patients with suspected celiac disease.

Prospective multicentre clinical study on inter- and intrapatient genetic variability for antimicrobial resistance of Helicobacter pylori.

OBJECTIVES: We report on a large prospective, multicentre clinical investigation on inter- and intrapatient genetic variability for antimicrobial resistance of Helicobacter pylori. METHODS: Therapy-naive patients (n = 2004) who had undergone routine diagnostic gastroscopy were prospectively included from all geographic regions of Austria. Gastric biopsy samples were collected separately from antrum and corpus. Samples were analysed by histopathology and real-time PCR for genotypic resistance to clarithromycin and quinolones. Clinical and demographic information was analysed in relation to resistance patterns. RESULTS: H. pylori infection was detected in 514 (26%) of 2004 patients by histopathology and confirmed in 465 (90%) of 514 patients by real-time PCR. PCR results were discordant for antrum and corpus in 27 (5%) of 514 patients, indicating inhomogeneous infections. Clarithromycin resistance rates were 17% (77/448) and 19% (84/455), and quinolone resistance rates were 12% (37/310) and 10% (32/334) in antrum and corpus samples, respectively. Combination of test results per patient yielded resistance rates of 21% (98/465) and 13% (50/383) for clarithromycin and quinolones, respectively. Overall, infection with both sensitive and resistant H. pylori was detected in 65 (14%) of 465 patients. CONCLUSIONS: Anatomically inhomogeneous infection with different, multiple H. pylori strains is common. Prospective clinical study design, collection of samples from multiple sites and microbiologic methods that allow the detection of coinfections are mandatory for collection of reliable data on antimicrobial resistance patterns in representative patient populations. (ClinicalTrials.gov identifier: NCT02925091).

Structural changes of sarcoplasmic reticulum Ca(2+)-ATPase upon Ca2+ binding studied by simultaneous measurement of infrared absorbance changes and changes of intrinsic protein fluorescence.

Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca2+ from the photolabile Ca2+ chelator 1-(2-nitro-4,5-dimethoxy)-N,N,N',N',- tetrakis[(oxycarbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1H2O and 2H2O were detected in the spectral region from 1800 cm-1 to 1200 cm-1, reflecting photolysis of DM-nitrophen and Ca2+ binding to the Ca(2+)-ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca2+ binding activity of the ATPase in this specific step. Ca2+ binding caused IR difference bands mainly in the region of amide I absorption of the polypeptide backbone, reflecting conformational changes of the protein. The small amplitude of the signals indicates that only a few residues perform local structural changes such as changes of bond angles or hydrogen bonding. Other absorbance changes appearing above 1700 cm-1 can be assigned to Ca2+ binding to Glu or Asp side chain carboxyl groups and concomitant deprotonation of these residues. This assignment is strengthened by downshifts of these bands by 4 cm-1 to 6 cm-1 upon 1H2O/2H2O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262-6267).

Two distinct rhodopsin molecules within the disc membrane of vertebrate rod outer segments.

The kinetics of the metarhodopsin I-II reaction have been measured over a wide range of temperatures (1-37C ) and pH values (4.5-8) with suspensions containing fragments of bovine rod outer segments. It was found that for all conditions the occurrence of metarhodopsin II could be described by two independent first-order processes. The fast component: slow component amplitude ratio depends upon pH and temperature. The kinetics of the lumi-metarhodopsin I reaction show the same pH dependence for the fast component: slow component amplitude ratio as the one observed for the metarhodopsin II signals. All the results observed could be described with the assumption that rhodopsin itself exists in two conformational states before bleaching which are in a pH and temperature-dependent equilibrium. This hypothesis is confirmed by its ability to explain some apparently anomalous observations in the literature.

Vibrational spectroscopy as a tool for probing protein function.

Vibrational spectroscopy has become increasingly important as a tool for understanding the mechanisms of photosystem II, phytochrome and terminal oxidases. More general enzymatic or receptor systems have been studied, opening a new field of applications. Femtosecond infrared pump/probe studies of the important amide-I band seem to provide a basis for its molecular and structural interpretation.

Distortion of the L-->M transition in the photocycle of the bacteriorhodopsin mutant D96N: a time-resolved step-scan FTIR investigation.

The D96N mutant of bacteriorhodopsin has often been taken as a model system to study the M intermediate of the wild type photocycle due to the long life time of the corresponding intermediate of the mutant. Using time-resolved step-scan FTIR spectroscopy in combination with a sample changing wheel we investigated the photocycle of the mutant with microsecond time resolution. Already after several microseconds an intermediate similar to the MN state is observed, which contrasts with the M state of the wild type protein. At reduced hydration M and N intermediates similar to those of wild type BR can be detected. These results have a bearing on the interpretation of the photocycle of this mutant. A mechanism is suggested for the fast rise of MN which provides some insight into the molecular events involved in triggering the opening of the cytosolic channel also of the wild type protein.

Asp85 is the only internal aspartic acid that gets protonated in the M intermediate and the purple-to-blue transition of bacteriorhodopsin. A solid-state 13C CP-MAS NMR investigation.

High-resolution solid-state 13C NMR spectra of the ground state and M intermediate of the bacteriorhodopsin mutant D96N with the isotope label at [4-13C]Asp and [11-13C]Trp were recorded. The NMR spectra show that Asp85 is protonated in the M intermediate. The environment of Asp85 is quite hydrophobic. On the other hand, Asp212 remains deprotonated and a slight shift to lower field indicates a more hydrophilic environment. Asp85 also protonates in the purple-to-blue transition of bacteriorhodopsin in the deionized membrane, where it experiences a similar environment to M. The shift of Trp resonances in M reflect a conformational change of the protein in forming the M intermediate.

Protonation changes during the photocycle of sensory rhodopsin II from Natronobacterium pharaonis.

The Fourier Transform Infrared (FTIR) spectra of photocycle intermediates of sensory rhodopsin II (pSRII) from Natronobacterium pharaonis were measured. The results of the FTIR experiments indicate considerable conformational movements of pSRII already at the stage of the early K-like intermediate which persist at least during the lifetime of the long lived intermediate. These changes in the amide bond region are more intense than those described for sensory rhodopsin I (SRI) and are quite similar to those observed for rhodopsin. Concomitantly with the deprotonation of the Schiff base a carboxyl group located in a hydrophobic environment is protonated. In analogy to bacteriorhodopsin, this carboxyl group might arise from Asp-75 which probably serves as counter ion to the Schiff base. The protonation reaction differs from the situation observed in SRI where the protonation is pH independent over the range of pH 5-8.

Z,E isomerization of the alpha-84 phycoviolobilin chromophore of phycoerythrocyanin from Mastigocladus laminosus investigated by Fourier-transform infrared difference spectroscopy.

The photoreaction of the phycoviolobilin (PVB) chromophore-containing part of phycoerythrocyanin (PEC) from Mastigocladus laminosus was investigated by Fourier-transform infrared spectroscopy (FT-IR). Difference spectra between the parent states P566 and P507 were obtained in 1H2O and 2H2O for the first time. The spectra are generally characterised by large changes in the range between 1710 and 1590 cm(-1) and by a strong difference band around 1270 cm(-1). In order to study the influence on the PVB chromophore upon aggregation, spectra of the alpha-subunit and the (alphabeta)3 trimer are compared, showing distinct differences which may be of relevance for the chromophore-protein and protein-protein interactions. The difference spectra demonstrate many similarities to the spectra recently obtained for the Pr --> meta-Rc transition of phytochrome [Foerstendorf et al. (1996) Biochemistry 35, 10793-10799]. In particular, a band around 1710 cm(-1), which was tentatively assigned to the C = O stretch of ring D is also observed in the spectra of PEC. It strongly supports this identification and the deduced molecular interpretation on the protonation state of the chromophore.

Evidence for the specific interaction of a lipid molecule with rhodopsin which is altered in the transition to the active state metarhodopsin II.

Comparing the FTIR difference spectra of the rhodopsin --> metarhodopsin II transition in membranes and in dodecylmaltoside detergent, characteristic variations are observed between 1715 and 1750 cm(-1). By repeating the measurements with the rhodopsin mutant D83N/E122Q, the spectral variation between the samples in membranes versus detergent could be assigned to a difference band at 1743(+)/1724(-) cm(-1), which does not exhibit a deuteration-induced downshift. We provide evidence that this band is probably caused by the C=O stretch of only one ester group of one lipid molecule. This group interacts with the dark state of rhodopsin, whereas in metarhodopsin II, the lipid molecule behaves as if it were in the bulk lipid phase.

Photoactivated state of rhodopsin and how it can form.

A variety of spectroscopic and biochemical studies of the photoreceptor rhodopsin have revealed conformation changes which occur upon its photoactivation. Assignment of these molecular alterations to specific regions in the receptor has been attempted by studying native opsin regenerated with synthetic retinal analogs or recombinant opsins regenerated with 11-cis retinal. We propose a model for the photoactivation mechanism which defines 'off' and 'on' states for individual molecular groups. These groups have been identified to undergo structural alterations during photoactivation. Analysis of mutant pigments in which specific groups are locked into their respective 'on' or 'off' states provides a framework to identify determinants of the active conformation as well as the minimal number of intramolecular transitions to switch to this conformation. The simple model proposed for the active-state of rhodopsin can be compared to structural models of its ground-state to localize chromophore-protein interactions that may be important in the photoactivation mechanism.

Conformational changes of cytosolic loops of bovine rhodopsin during the transition to metarhodopsin-II: an investigation by Fourier transform infrared difference spectroscopy.

In order to assign the structural changes of the protein, observed in the Fourier transform infrared (FT-IR) difference spectra of the rhodopsin-metarhodopsin-II transition, to specific regions of the protein, rhodopsin was treated by proteases. Nonilluminated and bleached rhodopsin was treated with protease K and papain. Rhodopsin digested in the bleached state was subsequently regenerated with 11-cis-retinal. From these modified samples the rhodopsin-metarhodopsin-II FT-IR difference spectra were measured. Comparing the difference spectra with that of unmodified rhodopsin, clear deviations in the amide-I and amide-II spectral range are observed. This indicates that in the unmodified pigment conformational changes of those parts of the cytosolic surface take place which are susceptible to the proteases. From the larger spectral changes obtained with samples digested in the bleached state it is concluded that the extent of modification is larger. The difference spectra of rhodopsin modified with 10 mM dithiothreitol support the existence of the 4th loop which also undergoes conformational changes. The spectral changes are interpreted in terms of a transition of an ordered structure of the loops in rhodopsin to a more random structure in metarhodopsin-II. The results demonstrate that by combining FT-IR spectroscopy with protein modification by specific proteases, conformational changes of the protein can be localized to specific regions.