Sodium-glucose cotransporter 2 inhibitors: a practical guide for the Dutch cardiologist based on real-world experience.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors include a relatively new class of glucose-lowering drugs that reduce plasma glucose concentrations by inhibiting proximal tubular reabsorption of glucose in the kidney, while increasing its excretion in urine. Recent large randomised controlled trials have demonstrated that many of these agents reduce the occurrence of major adverse cardiovascular events, hospitalisation for heart failure, cardiovascular death and/or chronic kidney disease progression in patients with and without type 2 diabetes mellitus (DM2). Given their unique insulin-independent mode of action and favourable efficacy and adverse-event profile, SGLT2 inhibitors are promising and they offer an interesting therapeutic approach for the cardiologist to incorporate into routine practice. However, despite accumulating data supporting this class of therapy, cardiologists infrequently prescribe SGLT2 inhibitors, potentially due to a lack of familiarity with their use and the reticence to change DM medication. Here, we provide an up-to-date practical guide highlighting important elements of treatment initiation based on real-world evidence and expert opinion. We describe how to change DM medication, including insulin dosing when appropriate, and how to anticipate any adverse events based on real-world experience in patients with DM2 in the Meander Medical Centre in Amersfoort, the Netherlands. This includes a simple algorithm showing how to initiate SGLT2 inhibitor treatment safely, while considering the consequence of the glucosuric effects of these inhibitors for the individual patient.

Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats.

AIMS: To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats. METHODS AND RESULTS: Germination was studied by feeding germ-free rats with spores of a B. thuringiensis strain harbouring a plasmid encoding green fluorescent protein (GFP), which enabled quantification of germinated bacteria by flow cytometry. To study in vivo conjugation, germ-free rats were first associated with a B. thuringiensis recipient strain and after 1 week an isogenic donor strain harbouring the conjugative plasmid pXO16 was introduced. Both strains were given as spores and transfer of pXO16 was observed from the donor to the recipient strain. CONCLUSIONS: Bacillus thuringiensis is able to have a full life cycle in the intestine of gnotobiotic rats including germination of spores, several cycles of growth and sporulation of vegetative cells. For the first time conjugative plasmid transfer in a mammalian intestinal tract was shown between two B. thuringiensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of B. thuringiensis are used worldwide to combat insect pests, and this study brings new insights into the nature of B. thuringiensis showing the potential of the bacteria to germinate and transfer DNA in the mammalian intestinal tract.

Influence of folate status and polyphenol intake on thiamin status of Irish women.

The relationship of folate status and polyphenol intake to thiamin status was studied in 80 randomly selected elderly and young Irish women, with key variables affecting thiamin nutrition controlled for. Folate, thiamin, and polyphenol intakes were measured during a 4-wk baseline (elderly and young) and 6-wk double-blind (elderly) supplementation period. Only elderly subjects were randomly assigned to placebo, folic acid (400 micrograms), thiamin (10 mg), or folic-acid-plus-thiamin groups. Thiamin status (TPP effect) was not affected by folate status (plasma and erythrocyte folate) during the baseline period or with folic acid supplementation alone. Polyphenol intake was not correlated with thiamin status. Only thiamin intake and thiamin supplementation significantly affected thiamin status. Because the majority of subjects (102 of 160) were initially thiamin deficient, enrichment of Irish grain products with thiamin is recommended.

An investigation of the centres of pressure under the foot while walking.

The forces under the foot while walking have been measured using a high sensitivity force-plate of the strip-suspended type combined with simultaneous filming of the sole of the foot. The recording of data and the calculation and plotting of results were much simplified by computer aid. Normal and abnormal feet, both barefoot and shod, were investigated in sixteen subjects. It was found that in normal barefoot walking the forefoot carried a total load of the order of three times that of the heel. When footwear was worn the function of the forefoot was progressively reduced as the rigidity of the sole of the shoe increased. Painful conditions of the forefoot also produced a large reduction in the proportion of the total load transferred.

Kinetics of conjugative transfer: a study of the plasmid pXO16 from Bacillus thuringiensis subsp. israelensis.

The aggregation-mediated conjugation system of Bacillus thuringiensis subsp. israelensis, encoded by the 200-kb plasmid pXO16, is highly potent in transferring itself and efficient in mobilizing other nonconjugative plasmids. The present study reveals some salient features of this conjugation system. Our observations can be summarized as follows: (i) The conjugative transfer takes about 3(1/2) to 4 min. For a 200-kb plasmid this corresponds to about 1 kb per second. (ii) The ability to transfer the plasmid seems to be evenly distributed among the donors. (iii) Functionally, the mating complex was found to consist of one donor and one recipient cell, even though aggregates comprising thousands of interconnected cells are formed. (iv) Having donated the plasmid, the donor needs a "period of recovery" of about 10 min before it can redonate the plasmid. (v) Secondary transfer, i.e., transfer from newly formed transconjugants, is delayed about 40 min. This maturation time exceeds the generation time, and it may indicate that to display donor activity, a surface protein (the aggregation substance) has to be uniformly incorporated into the cell wall. Lastly, we found that when the experiments were sufficiently short and when the recipient cells were in excess compared with the donors, the process of conjugation could be reasonably described by a kinetic model analogous to the Michaelis-Menten model for enzyme catalysis. This allowed us to estimate (vi) the maximal conjugation rate to be about 0.05 transconjugant per donor per minute, and (vii) the Km value, i.e., the concentration of recipient that results in half of the maximal conjugation rate, to be about 4 x 10(6) recipients/ml.

Influence of thiamin supplementation on the health and general well-being of an elderly Irish population with marginal thiamin deficiency.

The effect of thiamin supplementation on the health and general well-being of 80 randomly selected healthy elderly Irish women, from a population with marginal thiamin deficiency, was studied. Key variables affecting thiamin status were controlled. Weekly dietary intakes, subjective feelings, and activity assessments were measured during a 4-week baseline and 6-week double blind treatment period. Clinical assessments were performed during the last week of each period. For treatment, subjects were randomly assigned to either thiamin (10 mg daily) or placebo groups. Compared to baseline and placebo supplemented values, thiamin-supplemented women experienced significantly increased appetite, energy intake, body weight and general well-being, and decreased fatigue. Thiamin supplementation also tended to reduce daytime sleep time, improve sleep patterns, and increase activity. These data suggest that evaluation of thiamin status is indicated when nonspecific conditions such as anorexia, weight loss, fatigue, depression, and sleep disorders are present in elderly persons.

Mobilization of "nonmobilizable" plasmids by the aggregation-mediated conjugation system of Bacillus thuringiensis.

The aggregation-mediated conjugation system of Bacillus thuringiensis subsp, israelensis (Bti), encoded by the 200-kb plasmid pXO16, is highly potent in transferring itself and efficient in mobilizing other nonconjugative plasmids. In the present study we have analyzed the native Bacillus cereus plasmid pBC16. This plasmid has previously been shown to harbor a mob gene (ORF beta) and a locus functioning as an oriT site in plasmid pLS20-mediated conjugation in Bacillus subtilis. However, in the conjugation system of Bti we found that a derivative of pBC16 deleted for both these loci was mobilizable, although at a reduced frequency. Another derivative of pBC16, containing a deletion spanning the first half of the coding region of the mob gene, was found to be nearly as mobilizable as the intact pBC16, suggesting its dispensability in the transfer process. Other plasmids based on the theta-replicating origins, pAM beta 1, pLS20, ori43, ori44, and ori60, were also consistently mobilized in the conjugation system encoded by Bti plasmid pXO16. Analyzing the conjugation process by the use of scanning electron microscopy revealed the presence of connections between cells in the mating mixtures. These connections did not appear in monocultures of the donor strain or the recipient strain and may be conjugational junctions.

The aggregation-mediated conjugation system of Bacillus thuringiensis subsp. israelensis: host range and kinetics of transfer.

The aggregation-mediated conjugation system in Bacillus thuringiensis subsp. israelensis encoded on the plasmid pXO16 is characterized by the formation of aggregates when Agr+ and Agr- cells are socialized in exponential growth. Using the aggregation phenotypes, we have identified potential recipients of the aggregation-plasmid pXO16 among Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Bacillus sphaericus, and 24 subspecies of B. thuringiensis. We found 14 Agr- strains, i.e., potential recipients of the aggregation system encoded by plasmid pXO16. Five strains contained a conjugative apparatus of their own and were excluded from further examinations. To monitor the transfer of plasmid pXO16, we constructed a transposon insertion of the plasmid with Tn5401. The study of the plasmid transfer of pXO16::Tn5401 indicated the secretion of bacteriocins from both donor strain and recipient strains. Only one out of the nine strains examined was unable to receive the aggregation-plasmid pXO16 and express the aggregation phenotype and the conjugative abilities. It was found that the transfer of plasmid pXO16 to Bacillus thuringiensis subsp. israelensis Agr- strains was 100%. All recipients had acquired the aggregation-plasmid pXO16 and converted to the Agr+ phenotype.

Replication mechanism and sequence analysis of the replicon of pAW63, a conjugative plasmid from Bacillus thuringiensis.

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.