Neue Ansätze des molekularen Targetings bei der Philadelphia-Chromosom-positiven Leukämie

In Philadelphia Chromosome (Ph) positive ALL and CML the fusion between BCR and ABL leads to the BCR/ABL fusion proteins, which induces the leukemic phenotype because of the constitutive activation of multiple signaling pathways down-stream to the aberrant BCR/ABL fusion tyrosine kinase. Targeted inhibition of BCR/ABL by ABL-kinase inhibitors induces apoptosis in BCR/ABL transformed cells and leads to complete remission in Ph positive leukemia patients. However, a large portion of patients with advanced Ph+ leukemia relapse and acquire resistance. Kinase domain (KD) mutations interfering with inhibitor binding represent the major mechanism of acquired resistance in patients with Ph+ leukemia. Tetramerization of BCR/ABL through the N-terminal coiled-coil region (CC) of BCR is essential for the ABL-kinase activation. Targeting the CC-domain forces BCR/ABL into a monomeric conformation, reduces its kinase activity and increases the sensitivity for Imatinib. Here we show that i.) targeting the tetramerization by a peptide representing the Helix-2 of the CC efficiently reduced the autophosphorylation of both WT BCR/ABL and its mutants; ii.) Helix-2 inhibited the transformation potential of BCR/ABL independently of the presence of mutations; iii.) Helix-2 efficiently cooperated with Imatinib as revealed by their effects on the transformation potential and the factor-independence related to BCR/ABL with the exception of mutant T315I. These findings suggest that BCR/ABL harboring the T315I mutation have a transformation potential which is at least partially independent from its kinase activity. Targeted inhibition of BCR/ABL by small molecule inhibitors reverses the transformation potential of BCR/ABL. We definitively proved that targeting the tetramerization of BCR/ABL mediated by the N-terminal coiled-coil domain (CC) using competitive peptides, representing the Helix-2 of the CC, represents a valid therapeutic approach for treating Ph+ leukemia. To further develop competitive peptides for targeting BCR/ABL, we created a membrane permeable Helix-2 peptide (MPH-2) by fusing the Helix-2 peptide with a peptide transduction tag. In this study, we report that the MPH-2: (i) interacted with BCR/ABL in vivo; (ii) efficiently inhibited the autophosphorylation of BCR/ABL; (iii) suppressed the growth and viability of Ph+ leukemic cells; and (iv) was efficiently transduced into mononuclear cells (MNC) in an in vivo mouse model. The T315I mutation confers resistance against all actually approved ABL-kinase inhibitors and competitive peptides. It seems not only to decrease affinity for kinase inhibitors but to confer additional features to the leukemogenic potential of BCR/ABL. To determine the role of T315I in resistance to the inhibition of oligomerization and in the leukemogenic potential of BCR/ABL, we investigated its influence on loss-of-function mutants with regard to the capacity to mediate factor-independence. Thus we studied the effects of T315I on BCR/ABL mutants lacking functional domains in the BCR portion indispensable for the oncogenic activity of BCR/ABL such as the N-terminal coiled coil (CC), the tyrosine phosphorylation site Y177 and the serine/threonine kinase domain (ST), as well as on the ABL portion of BCR/ABL (#ABL-T315I) with or without the inhibitory SH3 (delta SH3-ABL) domain. Here we report that i.) T315I restored the capacity to mediate factor independence of oligomerization_deficient p185BCR/ABL; ii.) resistance of p185-T315I against inhibition of the oligomerization depends on the phosphorylation at Y177; iii.) autophosphorylation at Y177 is not affected by the oligomerization inhibition, but phosphorylation at Y177 of endogenous BCR parallels the effects of T315I; iv.) the effects of T315I are associated with an intact ABL_kinase activity; v.) the presence of T315I is associated with an increased ABL_kinase activity also in mutants unable to induce Y177 phosphorylation of endogenous BCR; vi.) there is no direct relationship between the ABL-kinase activity and the capacity to mediate factor_independence induced by T315I as revealed by the #ABL-T315I mutant, which was unable to induce Y177 phosphorylation of BCR only in the presence of the SH3 domain. In contrast to its physiological counterpart c-ABL, the BCR/ABL kinase is constitutively activated, inducing the leukemic phenotype. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl binding pocket in a process called “capping”, which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, BCR/ABL “escapes” this auto-inhibition. Allosteric inhibition by myristate “mimics”, such as GNF-2, is able to inhibit unmutated BCR/ABL, but not the BCR/ABL that harbors the “gatekeeper” mutation T315I. Here we investigated the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. We demonstrate that inhibition of oligomerization was able not only to increase the efficacy of GNF-2 on unmutated BCR/ABL, but also to overcome the resistance of BCR/ABL-T315I to allosteric inhibition. These results strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. Taken together these data suggest that the inhibition of tetramerization inhibits BCR/ABL-mediated transformation and can contribute to overcome Imatinib-resistance. The study provides the first evidence that an efficient peptide transduction system facilitates the employ-ment of competitive peptides to target the oligomerization interface of BCR/ABL in vivo. Further the data show that T315I confers additional leukemogenic activity to BCR/ABL, which might explain the clinical behavior of patients with BCR/ABL -T315I-positive blasts. In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors.In der Philadelphia Chromosome (Ph) positiven ALL und CML hat die Fusion von BCR und ABL die Bildung eines BCR/ABL Fusionsprotein zur Folge. Dieses Fusionsprotein ist für den leukämischen Phänotypen aufgrund der konstitutiven Aktivierung vieler Signalwege unterhalb/Downstream der veränderten BCR/ABL fusionierten Tyrosinkinase.Eine zielgerichtete Inhibierung von BCR/ABL mittels ABL-Kinase-Inhibitoren induziert Apoptose in BCR/ABL transformierten Zellen und hat eine komplette Remission in Ph+ Leukämie Patienten zur Folge. Eine große Anzahl an Patienten mit fortgeschrittener Ph+ Leukämie erleiden einen Rückfall und entwickeln Resistenzen. Die Mutation der Kinasedomäne verhindert die Bindung von Inhibitoren und stellt somit den häufigsten Mechanimus von erworbenen Resistenzen in Patienten mit Ph+ Leukämie. Die Tetramerizierung von BCR/ABL mittels der N-Terminalen coiled-coil region (CC) von BCR ist notwendig für die Aktivierung der ABl-Kinase. Das Targeting der CC-Domäne zwingt BCR/ABL in eine monomere Konformation, was zu einer Reduzierung der Kinasaktivität und einer erhöhten Imatinib-Sensitivität führt. Wir können zeigen, daß i) das Angreifen der Tetramerisierung mittels einem Peptide, welches die Helix2 der CC-Domäne repräsentiert, reduziert die Autophosphorylierung sowohl von WT BCR/ABL, als auch seinen Mutanten. ii) Die Helix-2 inhibiert unabhängig von vorkommenden Mutationen das transformierende Potential von BCR/ABL. iii) Aufgrund der Effekte auf das transformierende Potential und dem Faktor unabhängigen Wachstum von BCR/ABL, mit Ausnahme der T315I-Mutation, konnte eine effektive Kooperation zwischen Helix-2 und Imatinib gezeigt werden. Die gezielte Hemmung von BCR/ABL durch Inhibitoren in Form kleiner Moleküle macht das Potential zur Transformation von BCR/ABL rückgängig. Wir haben eindeutig bewiesen, dass das Zielen auf die, durch die N-terminale coiled-coil Domäne (CC) vermittelte, Tetramerisierung von BCR/ABL mithilfe kompetitiver Peptide, welche die Helix-2 von CC repräsentieren, ein wirkungsvoller therapeutischer Ansatz zur Behandlung der Ph+Leukämie ist. Um die kompetitiven Peptide zum Angriff auf BCR/ABL weiter zu entwickeln, erzeugten wir eine Membran permeables Helix-2 Peptid (MPH-2), indem wir die Helix-2 Peptide mit einem Peptid-Transduktions-Tag fusionierten. In dieser Studie berichten wir, dass MPH-2: (i) mit BCR/ABL in vivo interagierte; (ii) effizient die Autophosphorylierung von BCR/ABL hemmte; (iii) Wachstum und Viabilität von Ph+ leukämischen Zellen unterdrückte; und (iv) in einem in vivo Maus-Modell effizient in mononukleäre Zellen (MNC) transduziert wurde. Die T315I Mutation von BCR/ABL weist eine Resistenz gegen alle momentan sich in medizinischen Studien befindlichen ABL-kinase Inhibitoren und kompetitiven Peptiden auf. Es scheint nicht nur die Bindungsaffinität der Kinase Inhibitoren zu vermindern sondern erzeugt zusätzliche Eigenschaften, die das leukämogene Potential von BCR/ABL verstärken. Um die Rolle der T315I Mutation auf dessen fehlender Inhibition der für das leukämogene Potential wichtigen Oligomerisierung von BCR/ABL näher zu bestimmen, untersuchten wir seinen Einfluß auf zusätzliche “loss-of-function” Mutanten von BCR/ABL in bezug auf deren Fähigkeit eine Faktor-Unabhängigkeit zu erzeugen. Entsprechend erzeugten wir BCR/ABL Mutanten, denen notwendige funktionale Domänen bezüglich des onkogenen Potenzials fehlen. Im BCR-Anteil deletierten wir die N-terminale coiled coil (CC) Domäne, die Tyrosine Phosphorylierungs Stelle Y177 und die Serine/Threonine Kinase Domäne (ST) sowie im ABL_Anteil die inhibitorische SH3 (delta SH3-ABL) Domäne. Aus den vorliegenden Arbeiten ergaben sich folgende Ergebnisse i.) T315I stellt die fehlende Faktor Unabhängigkeit von hämatopoetischen Zellen mit Oligomerizations defizienten p185BCR/ABL Mutanten wieder her; ii.) Die Resistenz von p185-T315I gegen die Inhibition der Oligomerisation ist abhängig von der Phosphorylierung am Y177; iii.) Die Autophosphorylierung am Y177 wird durch die Inhibition der Oligomerisierung nicht beeinträchtigt, jedoch die Phosphorylierung am Y177 von endogenem BCR wird durch die T315I Mutation verstärkt; iv.) Die Effekte von T315I sind mit einer intakten ABL_Kinase Aktivität assoziiert; v.) Das Vorhandensein der T315I Mutation ist assoziiert mit einer verstärkten ABL_kinase Aktivität, welches sich auch in Mutanten zeigt, die die Y177 Phosphorylierung von endogenem BCR nicht induzieren; vi.) Es gibt keinen direkten Zusammenhang zwischen der ABL-Kinase Aktivität und der durch T315I induzierten Faktorunabhängigkeit. Im Gegensatz zu der ABL-T315I Mutante, die nur in der Anwesenheit der SH3 Domäne nicht in der Lage war eine Phosphorylierung am Y177 von endogenem BCR zu induzieren. Im Gegensatz zu seinem physiologischen Pendant c-Abl, ist die BCR/ABL Kinase, die den leukämischen Phänotyp induziert, konstitutiv aktiviert. Der N-Terminus von c-ABL (Cap region) wirkt an der Regulation seiner Kinasefunktion mit. Er ist myristoyliert und in einem Prozess, genannt „capping“ bindet der Myristinsäurerest an eine hydrophobe Tasche in der Kinasedomäne, bekannt als Myristinsäurebindungstasche. Daraus resultiert eine autoinhibitorische Konformation. Da die Cap-region durch den N-terminus von BCR/ABL ersetzt wurde, entgeht BCR/ABL dieser Autoinhibition. Allosterische Inhibition durch Myristeinimitatoren, wie GNF-2, sind in der Lage nicht mutiertes BCR/ABL zu inhibieren, nicht jedoch BCR/ABL, das die „Gatekeeper“-Mutation T315I beherbergt. Wir untersuchen hier die Möglichkeit die Effizienz der allosterischen Inhibtion durch Blockierung der BCR/ABL-Oligomerisierung zu erhöhen. Wir demonstrieren, dass die Inhibtion der Oligomerisierung nicht nur in der Lage war die Effizienz von GNF-2 auf das nicht mutierte BCR/ABL zu erhöhen, sondern auch die Resistenz von BCR/ABL-T315I zu überwinden hin zur allosterischen Hemmung. Diese Ergebnisse lassen stark annehmen, dass das Ansprechen der allosterischen Hemmung durch GNF-2 in umgekehrtem Verhältnis zum Grad der Oligomerisierung von BCR/ABL steht. Zusammengenommen deuten diese Daten auf eine Inhibierung der Tetramerizierung hin, welsche die BCR/ABL vermittelte Transformation hemmt. Diese führt zu einer Überwindung der Imatinib Resistenz. Diese Studie bietet erste Evidenz, dass ein effizientes Peptid-Transduktions-System die Anwendung von kompetitiven Peptiden, um auf die Oligomerisierungs-Schnittstelle von BCR/ABL zu zielen, in vivo unterstützt. Des weitern zeigen die Daten, daß T315I zusätzliche leukämische Aktivität von BCR/ABL induziert, welches eine mögliche Erklärung für klinische Prognose von Patienten mit BCR/ABL-T315I positiven Blasten darstellt. Zusammenfassend lässt sich sagen, dass unsere Beobachtungen einen neuen Ansatz für molekulare Angriffspunkte für BCR/ABL und seine resistenten Mutanten etablieren, bestehend aus der Kombination von Oligomerisierungs- und allosterischen Inhibitoren

Bacteria in biofuel production

In this work, bacteria are regarded either as production organisms or as interacting organisms with another production organism, namely yeast. The aim of using bacteria as a production organism was, to optimize Rubisco activity by introducing additional gene cassettes of Rubisco in Cyanobacteria. At first, RBC operon was tried to clone in pDF-Trc plasmid, using infusion cloning, but did not obtained E. coli transformants containing the RBC operon, when E.coli was transformed with infusion cloning reaction. This might be because of the plasmid concentration that was used, which was less than the recommended concentration. When cloning the RBC operon by conventional restriction/ligation, E. coli transformants containing the operon were obtained. Further characterisation of the cloned operon could not be performed during this project. In the second part of the project, the ethanol tolerant lactic acid bacterium Lactobacillus vini was investigated, with a special focus on its interaction with ethanol producing organisms, Saccharomyces cerevisiae and Dekkera bruxellensis. Lower ethanol concentrations did not have any considerable effect on the growth of L. vini. However, at higher ethanol concentration, a continuous decrease in cell numbers of L. vini was observed. The study shows, that L. vini can tolerate and survive higher ethanol concentrations, as compared to other microorganisms. L. vini had better growth rate, when it was co-cultured separately with Saccharomyces cerevisiae and Dekkera bruxellensis compared to, when it was co-cultured with both the yeasts. Ethanol production in batch was relatively higher when L. vini was co-cultured with S. cerevisiae compared to co-cultivation with D. bruxellensis and with both yeasts, which is most probably due to the rapid growth of S. cerevisiae compared to D. bruxellensis

p185(BCR/ABL) has a lower sensitivity than p210(BCR/ABL) to the allosteric inhibitor GNF-2 in Philadelphia chromosome-positive acute lymphatic leukemia

Background: The t(9;22) translocation leads to the formation of the chimeric breakpoint cluster region/c-abl oncogene 1 (BCR/ABL) fusion gene on der22, the Philadelphia chromosome. The p185(BCR/ABL) or the p210(BCR/ABL) fusion proteins are encoded as a result of the translocation, depending on whether a "minor" or "major" breakpoint occurs, respectively. Both p185(BCR/ABL) and p210(BCR/ABL) exhibit constitutively activated ABL kinase activity. Through fusion to BCR the ABL kinase in p185(BCR/ABL) and p210(BCR/ABL) "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. A novel class of compounds including GNF-2 restores allosteric inhibition of the kinase activity and the transformation potential of BCR/ABL. Here we investigated whether there are differences between p185(BCR/ABL) and p210(BCR/ABL) regarding their sensitivity towards allosteric inhibition by GNF-2 in models of Philadelphia chromosome-positive acute lymphatic leukemia. Design and methods: We investigated the anti-proliferative activity of GNF-2 in different Philadelphia chromosome-positive acute lymphatic leukemia models, such as cell lines, patient-derived long-term cultures and factor-dependent lymphatic Ba/F3 cells expressing either p185(BCR/ABL) or p210(BCR/ABL) and their resistance mutants. Results: The inhibitory effects of GNF-2 differed constantly between p185(BCR/ABL) and p210(BCR/ABL) expressing cells. In all three Philadelphia chromosome-positive acute lymphatic leukemia models, p210(BCR/ABL)-transformed cells were more sensitive to GNF-2 than were p185BCR/ABL-positive cells. Similar results were obtained for p185(BCR/ABL) and the p210(BCR/ABL) harboring resistance mutations. Conclusions: Our data provide the first evidence of a differential response of p185(BCR/ABL)- and p210(BCR/ABL)- transformed cells to allosteric inhibition by GNF-2, which is of importance for the treatment of patients with Philadelphia chromosome-positive acute lymphatic leukemia

Exploring the tension between the discourses of affirmative action and the knowledge economy

Thesis (MEd)--Stellenbosch University, 2013.BibliographyENGLISH ABSTRACT: South Africa needs to ensure equal opportunity for all to higher education, and given that it also needs to correct the drastic imbalances brought about by apartheid, affirmative action is seen as a strategy to pursue both goals. Affirmative action is comprised of programs and policies that grant favorable treatment on the basis of race or gender to government-defined “disadvantaged” individuals. However, affirmative action is not without its own challenges and difficulties. The main question that this thesis addresses is “what are the tensions between applying affirmative action policies in South African higher education institutions and the demands of a knowledge economy within a globalised world?” I argue that though universities need to be more demographically representative and broaden access to previously disadvantaged individuals by adjusting entry requirements, they cannot compromise on their quality of graduates by adjusting their exit criteria in line with racial representivity. That would undermine the very worth of higher education as a social good, the dignity of the individual graduate, as well as the economic growth of the country. Accusations that affirmative action is merely “reverse discrimination” are refuted by an appeal to Rawls’s Principle of Difference which holds that policies of inequality can be socially just. Drawing on Charles Taylor and Wally Morrow, I posit that within a democracy, affirmative action should be seen as a shared rather than a convergent good for broadening access to quality education. But whereas broadening formal access seems like a legitimate and necessary step to address the inherited inequities, the broadening of epistemological access would undermine the very aims of quality education. Furthermore, I argue that formal access should be driven by the politics of difference, but that epistemological access that ensures educational success should be driven by the politics of equal dignity. In order to see how some of these concepts and policies of affirmative action play out in an actual institution, I look at the University of Cape Town (UCT). Here the main debates relating to its affirmative action policy are whether demographic representivity is the only outcome for evaluating the success of affirmative action, and whether “disadvantaged” individuals should be selected on criteria other than race. It also considers whether its affirmative action policies could compromise its functioning and ability to supply quality qualifications to the required number of disadvantaged individuals. There is no easy and simple answer to whether affirmative action in fact promotes equal opportunity to higher education and equips all South African graduates with the necessary skills for a knowledge economy. It would be therefore important to do further research on what nonrace based affirmative action policies might entail while keeping in mind the shifts in the global economy and the need for academic rigor. Furthermore, more longitudinal research needs to be done on the complex consequences of affirmative action, on both an individual level with issues of identity and career mobility, and on a broader socio-economic level with issues of economic growth and social welfare.AFRIKAANSE OPSOMMING: Suid-Afrika moet hom beywer tot die daarstelling van gelyke geleenthede vir almal tot hoëronderwys, en gegewe dat daar ’n behoefte is om drastiese ongelykhede van apartheid reg te stel, word regstellende aksie gesien as a strategie om beide doelstellings na te streef. Regstellende aksie bestaan uit programme en beleide wat daarop gemik is om begunstigde behandeling te dien aan “voorheen benadeelde” individue, soos deur die staat gedefineer, op grond van ras en geslag. Maar regstellende aksie is nie sonder sy eie uitdagings en swaarhede nie. Die hoofvraag wat hierdie tesis addreseer, is: “Watter gespannenhede is daar tussen die uitvoering van regstellende aksie beleide in Suid-Afrikaanse Hoëronderwys instellings en die eise van ’n kennis-ekonomie binne ’n geglobaliseerde wêreld?” Ek argumenteer dat, ofskoon daar ’n behoefte is vir universiteite om meer demografies verteenwoordigend te wees en hul toegang tot voorheen benadeelde individue te verbreed deur toelatingsvereistes te wysig, kan hulle nie kompromeer op hul gehalte van gegradueerdes deur uitgangskriteria in lyn met ras verteenwoordiging nie. Dit sal juis die waarde van hoëronderwys as ’n sosiale goedheid, die waardigheid van die individule gegradueerde asook die ekonomiese groei van die land ondermyn. Aantygings dat regstellende aksie bloot “wedergekeerde diskriminasie” is, word weerlê deur ’n verwysing na Rawls se Beginsel van Verskil wat stel dat beleide van ongelykhede maatskaplike regverdiging kan hê. Gegrond op Charles Taylor en Wally Morrow, postuleer ek dat, binne ’n demokrasie, regstellende aksie beskou moet word as ’n gedeelde eerder as ’n konvergente goedheid om gehalte onderwys verder toeganklik te maak. Maar waar verbrede formele toegang gesien kan word as ’n wettige en nodige stap om geërfde ongelykhede aan te spreek, sal die verbreding van epistemologiese toegang juis die doelstellings van gehalte onderwys ondermyn. Verder voer ek aan dat formele toegang aangedryf moet word deur die politiek van verskil, maar dat epistemologiese toegang wat opvoedkundige sukses verseker, aangedryf moet word deur die politiek van gelyke waardigheid. Ten einde te sien hoe van hierdie konsepte en beleide van regstellende aksie hulself uitspeel in eintlike inrigtings van onderwys, kyk ek na die Universiteit Kaapstad (UK). Hier draai die debat aangaande regstellende aksie beleid om of die demografiese verteenwoordiging die enigste uitkoms is ter evaluering van die sukses van regstellende aksie, en of “benadeelde” individue geselekteer moet word op grond van kriteria anders as ras. Dit (UK) oorweeg ook of sy regstellende beleide sy funksionering en vermoë om gehalte kwalifikasies aan die verlangde getal benadeelde individue kompromiteer. Daar is geen eenvoudige en maklike antwoord betreffende regstellende aksie en of dit gelyke geleenthede tot hoëronderwys promoveer en alle Suid-Afrikaanse gegradueerders toerus met die nodige bevoegdhede vir ’n kennis-ekonomie nie. Dit sal derhalwe belangrik wees om verdere navorsing te doen oor wat nie-rasgebaseerde regstellende aksie kan behels terwyl in gedagte gehou word die skuiwe in die globale ekonomie en die behoefte aan akademiese kwaliteit. Verder moet veel meer longitudinale navorsing gedoen word oor die ingewikkelde gevolge van regstellende aksie op beide die individuele vlak met kwessies van identiteit en beroepsmobiliteit en op breër sosio-ekonomiese vlak met kwessies van ekonomiese groei en maatskaaplike welsyn

Protecting tear-film stability under adverse environmental conditions using a mucomimetic with a non-Newtonian viscosity agent

Background and Objectives: Tamarind-seed polysaccharide (TSP) and hyaluronic acid (HA) have mucoadhesive properties that improve drug absorption and delay in drug elimination from the ocular surface. We aimed to evaluate TSP/HA-containing formulation for its efficiency in dry-eye symptoms induced by adverse environments and the interaction between mucomimic polymer and tear-film parameters. Materials and Methods: The participants were exposed to 5% relative humidity (RH) in a Controlled Environment Chamber (CEC) under constant room temperature (21 °C). Tear-film parameters were assessed at 40% RH and 5% RH. Rohto Dry Eye Relief drops were used in the two treatment modalities, protection (drops instilled before exposure to the dry environment) and relief (drops instilled after exposure to the dry environment). The HIRCAL grid, Servomed EP3 Evaporimeter, and Keeler’s TearScope-Plus were used to screen for non-invasive tear break-up time (NITBUT), tear evaporation rate, and lipid-layer thickness (LLT) using protection and relief treatment methodology. Results: LLT was found to be significantly thinner at 5% RH compared with at 40% RH (p = 0.007). The median LLT dropped from 50–70 nm (grade 3) at 40% RH to 10–50 nm (grade 2) at 5% RH. TSP/HA eye drops significantly augment LLT in both treatment modalities, protection (p = 0.01) and relief (p = 0.004) at 5% RH. The mean evaporation rate doubled from 40.93 at 40% RH to 82.42 g/m2/h after exposure to 5% RH. In protection mode, the TSP/HA allowed the average evaporation rate to be much lower than when no TSP/HA was used at 5% RH (p &lt; 0.008). No alteration in evaporation rate was recorded when the TSP/HA drop was used after exposure (relief). The mean NITBUT was reduced from 13 s in normal conditions to 6 s in the dry environment. Instillation of TSP/HA eye drops resulted in significant improvement (p = 0.006) in tear stability, where the NITBUT increased to 8 s in both protection (before exposure) and relief (after exposure) (p = 0.001). Although improved, these values were still significantly lower than NITBUT observed at 40% RH. Conclusions: Significant protection of tear-film parameters was recorded post instillation of TSP/HA eye drop under a desiccating environment. Both treatment methods (protection and relief) were shown to be effective. The presence of TSP/HA enhances the effectiveness of teardrops in protecting the tear-film parameters when exposed to adverse environments.</p

Does Caesalpinia bonducella ameliorate genotoxicity? An in vitro study in human lymphocyte culture and in vivo study in Albino mice

AbstractMost of the world’s populations residing in developing countries depend on alternative medicine and use of plant ingredients. The plant Caesalpinia bonducella belongs to the family of Caesalpiniaceae and it is commonly known as Natakaranja in Hindi. It contains bonducin and two phytosterols namely sitosterol and hepatsane. The twigs and young leaves of C. bonducella are rationally used for curing tumors, inflammation and liver disorders. In our present work alcoholic extracts of this ayurvedic plant were tested for their antimutagenic and anticarcinogenic properties. The aim of the study is to investigate the antimutagenic and antigenotoxic potential of alcoholic extracts of C. bonducella against methyl methane sulfonate (MMS) induced genotoxicity. In this experiment we have used in vitro method i.e., human lymphocyte culture and in vivo method in bone marrow cells of albino mice, while the parameters studied included chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and cell growth kinetics (RI) both in the presence as well as in the absence of exogenous metabolic activation system for in vitro studies whereas total aberrant cells and the frequencies of aberrations were used for in vivo methods. Alcoholic extracts of C. bonducella significantly reduce chromosomal aberration from 42.75%, 44.25%, and 51.75% levels [at 24, 48, and 72h due to methyl methane sulfonate (MMS)] to 28.50%, 30.25%, and 35.10%, respectively similarly sister chromatid exchanges were reduced from 7.70±1.50 to 5.20±1.50 at 48h of treatment and replication index was enhanced in vitro for each concentration and duration of treatment and their ameliorating potential was dose and duration dependent. Similarly these extracts significantly reduced the number of aberrant cells and frequency of aberrations per cell in in vivo

Healthcare resilience to extreme events: A hospital staff perspective

The coronavirus (COVID-19) pandemic demonstrated how vulnerable and unprepared most healthcare sectors are to major disasters. Several studies have been published reporting factors that affect staff attendance during extreme events. However, these factors are limited and do not provide a full picture of why staff do or do not attend workplaces during major emergencies nor the impact of staff absences on healthcare service delivery. This study presents the factors influencing staff attendance during an extreme event and the impact staff attendance has on the continuity of healthcare services in one of the several independent British Isles hospitals. This study highlights that staff attendance depends on many contributors such as workload, stress, motivation, proximity of work to home, transportation networks, and dependents. The absence of any staff member, despite their role, level, or background, will have an impact on the functionality of a hospital. The study concludes that staff absence severely impedes the continuity of healthcare service, impacting services that provide ventilators and other essential services required during extreme events such as the COVID-19 pandemic and extreme weather events