Survey on the use of buprenorphine patches in the palliative care practice

Transdermal buprenorphine is a new formulation of the old drug available for the treatment of cancer and non-cancer pain. The drug offers number of interesting new features and was found effective in clinical trials in cancer patients with pain. We performed a survey of the use of buprenorphine patches for one year. In the survey we included 58 admitted patients (67 admission periods), whose clinical records and drug charts were subjected to analysis. Opioid naive patients were started either on 5 or 10 μg/hour. Mean buprenorphine dose was 22.3 μg/hour (95% CI: 16–28.6), increased on day 8 to 25.4 μg/hour (95% CI: 18.6–32) and ended up at the dose of 31.3 μg/hour (95% CI: 20.9–41.6) on the last day of treatment; day 19 (95% CI: 14.5–23.5). The overall dose increase was approximately 2% per day. Approximately half of the patients needed beside buprenorphine other opioids either in a slow release or immediate release form, usually morphine or oxycodone. Swapping from morphine, oxycodone and fentanyl to buprenorphine was without problems in all of the cases. The doses of all opioids administered calculated as oral morphine equivalents showed insignificant decreases for morphine and oxycodone to buprenorphine swaps. In case of fentanyl the oral morphine equivalents of opioids were significantly lower after swap (p = 0.0039). No signs of antagonism between the drugs were observed. In conclusion: buprenorphine patches appear to be useful in the treatment of cancer pain, either as monotherapy or in combination with other opioids. Swap from fentanyl to buprenorphine offers perspective of achievement of pain control with much less toxicity and should be investigated in more detail. Adv. Pall. Med. 2010; 9, 2: 39–44Transdermal buprenorphine is a new formulation of the old drug available for the treatment of cancer and non-cancer pain. The drug offers number of interesting new features and was found effective in clinical trials in cancer patients with pain. We performed a survey of the use of buprenorphine patches for one year. In the survey we included 58 admitted patients (67 admission periods), whose clinical records and drug charts were subjected to analysis. Opioid naive patients were started either on 5 or 10 μg/hour. Mean buprenorphine dose was 22.3 μg/hour (95% CI: 16–28.6), increased on day 8 to 25.4 μg/hour (95% CI: 18.6–32) and ended up at the dose of 31.3 μg/hour (95% CI: 20.9–41.6) on the last day of treatment; day 19 (95% CI: 14.5–23.5). The overall dose increase was approximately 2% per day. Approximately half of the patients needed beside buprenorphine other opioids either in a slow release or immediate release form, usually morphine or oxycodone. Swapping from morphine, oxycodone and fentanyl to buprenorphine was without problems in all of the cases. The doses of all opioids administered calculated as oral morphine equivalents showed insignificant decreases for morphine and oxycodone to buprenorphine swaps. In case of fentanyl the oral morphine equivalents of opioids were significantly lower after swap (p = 0.0039). No signs of antagonism between the drugs were observed. In conclusion: buprenorphine patches appear to be useful in the treatment of cancer pain, either as monotherapy or in combination with other opioids. Swap from fentanyl to buprenorphine offers perspective of achievement of pain control with much less toxicity and should be investigated in more detail. Adv. Pall. Med. 2010; 9, 2: 39–4

Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

<p>Abstract</p> <p>Background</p> <p>Restriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a <it>Thermus </it>sp. enzyme family, which belongs to type II restriction endonucleases (REases), however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II.</p> <p>Results</p> <p>Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the <it>tspgwi </it>gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the <it>Thermus </it>sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase) and methyltransferase (MTase) activities of wild type (wt) TspGWI (either recombinant or isolated from <it>Thermus </it>sp.) are dependent on the presence of divalent cations.</p> <p>Conclusion</p> <p>TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/E)XK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module of the HsdR subunit and the additional domains that are involved in subunit-subunit interactions in Type I systems. The MTase and REase activities of TspGWI are autonomous and can be uncoupled. Structurally and functionally, the TspGWI protomer appears to be a streamlined 'half' of a Type I enzyme.</p

Badanie dotyczące stosowania buprenorfiny w plastrach w opiece paliatywnej

Przezskórne podawanie buprenorfiny jest nową formą starego leku dostępną w terapii bólu nowotworowego i nienowotworowego. Lek ten ma liczne zachęcające właściwości i okazał się skuteczny w badaniach klinicznych u chorych na nowotwory, cierpiących z powodu bólu. Autorzy niniejszej pracy przeprowadzili badanie dotyczące stosowania buprenorfiny w plastrach przez okres 1 roku. Do badania włączono 58 chorych (67 okresów hospitalizacji), analizowano ich historie chorób i karty leków. Chorzy niestosujący wcześniej opioidów zaczynali od dawki 5 lub 10 &#956;g/h. Średnia dawka buprenorfiny wynosiła 22,3 &#956;g/h [95% CI: 16-28,6], zwiększana w 8. dniu do 25,4 &#956;g/h (95% CI: 18,6-32), aż do ostatecznej dawki 31,3 &#956;g/h (95% CI: 20,9-41,6) w ostatnim, 19. dniu leczenia (95% CI: 14,5-23,5). Łączny wzrost dawki wynosił średnio 2% dziennie. Około połowa chorych wymagała, oprócz stosowania buprenorfiny, podania innych opioidów o powolnym lub natychmiastowym uwalnianiu - zwykle były to morfina lub oksykodon. Zamiana z morfiny, oksykodonu czy fentanylu na buprenorfinę przebiegała bez problemów u wszystkich chorych. Wykazano nieistotny statystycznie spadek zużycia morfiny czy oksykodonu przy zamianie na buprenorfinę, w przeliczeniu na ekwiwalenty doustnej morfiny. W przypadku fentanylu dawka liczona jako ekwiwalenty doustnej morfiny była istotnie mniejsza (p = 0,0039) po zamianie na buprenorfinę. Nie stwierdzono antagonistycznych działań między lekami. Podsumowując, buprenorfina w plastrach wydaje się skuteczna w terapii bólu nowotworowego, zarówno w monoterapii, jak i w leczeniu skojarzonym z innymi opioidami. Zamiana z fentanylu może być alternatywą dla osiągnięcia lepszej kontroli bólu ze znacznie mniejszą toksycznością - zagadnienie to powinno być dokładniej zbadane. Medycyna Paliatywna w Praktyce 2010; 4, 3: 99-10

Modified ‘one amino acid-one codon’ engineering of high GC content TaqII-coding gene from thermophilic Thermus aquaticus results in radical expression increase

BACKGROUND: An industrial approach to protein production demands maximization of cloned gene expression, balanced with the recombinant host’s viability. Expression of toxic genes from thermophiles poses particular difficulties due to high GC content, mRNA secondary structures, rare codon usage and impairing the host’s coding plasmid replication. TaqII belongs to a family of bifunctional enzymes, which are a fusion of the restriction endonuclease (REase) and methyltransferase (MTase) activities in a single polypeptide. The family contains thermostable REases with distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI and a few enzymes found in mesophiles. While not being isoschizomers, the enzymes exhibit amino acid (aa) sequence homologies, having molecular sizes of ~120 kDa share common modular architecture, resemble Type-I enzymes, cleave DNA 11/9 nt from the recognition sites, their activity is affected by S-adenosylmethionine (SAM). RESULTS: We describe the taqIIRM gene design, cloning and expression of the prototype TaqII. The enzyme amount in natural hosts is extremely low. To improve expression of the taqIIRM gene in Escherichia coli (E. coli), we designed and cloned a fully synthetic, low GC content, low mRNA secondary structure taqIIRM, codon-optimized gene under a bacteriophage lambda (λ) P( R ) promoter. Codon usage based on a modified ‘one amino acid–one codon’ strategy, weighted towards low GC content codons, resulted in approximately 10-fold higher expression of the synthetic gene. 718 codons of total 1105 were changed, comprising 65% of the taqIIRM gene. The reason for we choose a less effective strategy rather than a resulting in high expression yields ‘codon randomization’ strategy, was intentional, sub-optimal TaqII in vivo production, in order to decrease the high ‘toxicity’ of the REase-MTase protein. CONCLUSIONS: Recombinant wt and synthetic taqIIRM gene were cloned and expressed in E. coli. The modified ‘one amino acid–one codon’ method tuned for thermophile-coded genes was applied to obtain overexpression of the ‘toxic’ taqIIRM gene. The method appears suited for industrial production of thermostable ‘toxic’ enzymes in E. coli. This novel variant of the method biased toward increasing a gene’s AT content may provide economic benefits for industrial applications

DNA-FACE™ - An <em>Escherichia coli</em>-based DNA Amplification-Expression Technology for Automatic Assembly of Concatemeric ORFs and Proteins

DNA-FACE™ (DNA Fragment Amplification & Concatemeric Expressed Nucleic Acids and Proteins) is a universal biotechnological platform, developed as Escherichia coli (E. coli) system. It is based on the ordered, head-to-tail directional ligation of the amplified DNA fragments. The technology enables the construction of targeted biomolecules - genetically programmed, concatemeric DNA, RNA, and proteins, designed to fit a particular task. The constructed, “artificial” (never seen in Nature) tandem repeat macromolecules, with specialized functions, may contain up to 500 copies of monomeric units. The technology greatly exceeds the current capabilities of chemical gene synthesis. The vector-enzymatic DNA fragment amplification assembles the DNA segments, forming continuous Open Reading Frames (ORFs). The obtained ORFs are ready for high-level expression in E. coli without a need for subcloning. The presented method has potential applications in pharmaceutical industry and tissue engineering, including vaccines, biological drugs, drug delivery systems, mass-production of peptide-derived biomaterials, industrial and environmental processes. The technology has been patented worldwide and used successfully in the construction of anti-HBV vaccines, pro-regenerative biological drugs and, recently, the anti-SARS-CoV-2 vaccine. The anti-SARS-CoV-2 vaccine, developed using the DNA-FACE™ technology, is nontoxic and induces strong immunological response to recombinant human spike and nucleocapsid proteins, as shown in animal studies

High-precision measurement of the half-life of 62^{62}Ga

The beta-decay half-life of 62Ga has been studied with high precision using on-line mass separated samples. The decay of 62Ga which is dominated by a 0+ to 0+ transition to the ground state of 62Zn yields a half-life of T_{1/2} = 116.19(4) ms. This result is more precise than any previous measurement by about a factor of four or more. The present value is in agreement with older literature values, but slightly disagrees with a recent measurement. We determine an error weighted average value of all experimental half-lives of 116.18(4) ms.Comment: 9 pages, 5 figures, accepted for publication in PR

Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities

<p>Abstract</p> <p>Background</p> <p>We previously defined a family of restriction endonucleases (REases) from <it>Thermus </it>sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI.</p> <p>Results</p> <p>TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the <it>Thermus </it>sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases.</p> <p>Conclusions</p> <p>TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.</p

Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development.

Early mouse development is accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2), which is essential for embryonic development. Here we show that genome-wide accumulation of H3K9me2 is crucial for postimplantation development, and coincides with redistribution of enhancer of zeste homolog 2 (EZH2)-dependent histone H3 lysine 27 trimethylation (H3K27me3). Loss of G9a or EZH2 results in upregulation of distinct gene sets involved in cell cycle regulation, germline development and embryogenesis. Notably, the H3K9me2 modification extends to active enhancer elements where it promotes developmentally-linked gene silencing and directly marks promoters and gene bodies. This epigenetic mechanism is important for priming gene regulatory networks for critical cell fate decisions in rapidly proliferating postimplantation epiblast cells.Wellcome Trust: Jan J Zylicz, Ufuk Günesdogan, Jamie A Hackett, Delphine Cougot, Caroline Lee, MA Surani, WT096738; European Commission (EC): Ufuk Günesdogan; Wellcome Trust: Jan J Zylicz, RG44593This is the final version of the article. It was first available from eLife via http://dx.doi.org/10.7554/eLife.0957

Calf thymus Hsc70 protein protects and reactivates prokaryotic and eukaryotic enzymes.

The heat-shock 70 protein (Hsp70) chaperone family is very conserved and its prokaryotic homologue, the DnaK protein, is assumed to form one of the cellular systems for the prevention and restoration of heat-induced protein denaturation. By using anti-DnaK antibodies we purified the DnaK homologue heat-shock cognate protein (Hsc70) from calf thymus to apparent homogeneity. This protein was classified as an eukaryotic Hsc70, since (i) monoclonal antibodies against eukaryotic Hsc70 recognized it, (ii) its amino-terminal sequence showed strong homology to Hsp70s from eukaryotes and, (iii) it had an intrinsic weak ATPase activity that was stimulated by various peptide substrates. We show that this calf thymus Hsc70 protein protected calf thymus DNA polymerases alpha and epsilon as well as Escherichia coli DNA polymerase III and RNA polymerase from heat inactivation and could reactivate these heat-inactivated enzymes in an ATP-hydrolysis dependent manner, likely leading to the dissociation of aggregates formed during heat inactivation. In contrast to this, DnaK protein was exclusively able to protect and to reactivate the enzymes from E.coli but not from eukaryotic cells. Finally, the addition of calf thymus DnaJ co-chaperone homologue reduced the amount of Hsc70 required for reactivation at least 10-fold

Parents’ concepts of the successful school child in seven Western cultures

Although children’s school success is a parental goal in most cultures, there is wide cultural variation in the qualities that parents most wish their children to develop for that purpose. A questionnaire contained forty-one child qualities was administered to 757 parents in seven cultural communities in Australia, Italy, theNetherlands, Poland, Spain, Sweden, and theUnited States. Exploratory factor analysis was conducted separately within each sample and results revealed both similarities and differences across the seven samples. The factor structures showed considerable similarity: four domains of characteristics (Cognitive Qualities, Social Qualities, Negative temperament, and Good Characters) were identified in each sample as strongly influencing children’s success in school. However, parents differed across the seven cultural communities in the importance they attributed to these factors. The results also reveal some culturally unique patterns in parents’ concepts of the successful schoolchild; the seven samples were differentiated by distinctive associations of individual qualities around the four common domains. These results offer new insights for incorporating perspectives from other cultures into our own concepts of what qualities are most important for children’s success in school, and how educators can be cognizant of differing cultural perspectives represented by the families whose children are their students