Outcomes of WHO-conforming, longer, all-oral multidrug-resistant TB regimens and analysis implications.

BACKGROUND: Evidence of the effectiveness of the WHO-recommended design of longer individualized regimens for multidrug- or rifampicin-resistant TB (MDR/RR-TB) is limited.OBJECTIVES: To report end-of-treatment outcomes for MDR/RR-TB patients from a 2015-2018 multi-country cohort that received a regimen consistent with current 2022 WHO updated recommendations and describe the complexities of comparing regimens.METHODS: We analyzed a subset of participants from the endTB Observational Study who initiated a longer MDR/RR-TB regimen that was consistent with subsequent 2022 WHO guidance on regimen design for longer treatments. We excluded individuals who received an injectable agent or who received fewer than four likely effective drugs.RESULTS: Of the 759 participants analyzed, 607 (80.0%, 95% CI 77.0-82.7) experienced successful end-of-treatment outcomes. The frequency of success was high across groups, whether stratified on number of Group A drugs or fluoroquinolone resistance, and ranged from 72.1% to 90.0%. Regimens were highly variable regarding composition and the duration of individual drugs.CONCLUSIONS: Longer, all-oral, individualized regimens that were consistent with 2022 WHO guidance on regimen design had high frequencies of treatment success. Heterogeneous regimen compositions and drug durations precluded meaningful comparisons. Future research should examine which combinations of drugs maximize safety/tolerability and effectiveness.

[Physiological changes during pregnancy]. / Physiologische Veränderungen in der Schwangerschaft.

The physiological state of a woman experiences multiple changes in the body during pregnancy. These alterations could be of particular importance in the medical care of pregnant women. This review article highlights the physiological developments of various organ systems throughout gestation with a focus on endocrinology, the cardiovascular system, hematology, the respiratory system and water balance.

Comparison of major bleeding risk in patients with non-valvular atrial fibrillation receiving direct oral anticoagulants in the real-world setting: a network meta-analysis.

OBJECTIVE: To conduct a systematic literature review (SLR) and network meta-analysis (NMA) of real-world studies comparing major bleeding risk among patients with non-valvular atrial fibrillation (NVAF) on direct oral anticoagulants (DOACs) or warfarin. METHODS: Systematic searches were conducted in MEDLINE and Embase for full-text articles published between January 1, 2003 and March 18, 2017. Eligible studies compared at least two of the following in a real-world setting: warfarin, apixaban, dabigatran, rivaroxaban, or edoxaban. A Bayesian NMA was conducted to estimate hazard ratios (HRs) for major bleeding using a random-effects model. RESULTS: Eleven studies were included in the NMA. Nine studies included DOACs vs Warfarin comparisons, and four studies included DOACs vs DOACs comparisons (two studies included both comparisons). Median follow-up duration ranged from 2.6-31.2 months. No evidence was identified for edoxaban. Apixaban was associated with a significantly lower risk of major bleeding compared to other oral anticoagulants (warfarin HR = 0.58; 95% credible interval [CrI] = 0.48-0.69; dabigatran = 0.73; 0.61-0.87; rivaroxaban = 0.55; 0.46-0.66). Dabigatran was associated with a significantly lower risk than warfarin (0.79; 0.71-0.88) and rivaroxaban (0.76; 0.67-0.85), and rivaroxaban was not statistically different from warfarin (1.05; 0.91-1.19). Sensitivity analyses with standard dose and sponsorship showed consistent results. CONCLUSION: DOACs were associated with lower or similar risk of major bleeding compared with warfarin in NVAF patients. Apixaban was associated with a significantly lower risk of major bleeding than other DOACs. Dabigatran was associated with a significantly lower risk of major bleeding compared to rivaroxaban and warfarin.

Risk of major bleeding in patients with non-valvular atrial fibrillation treated with oral anticoagulants: a systematic review of real-world observational studies.

OBJECTIVE: To conduct a systematic review of real-world (RWD) studies comparing the risk of major bleeding (MB) among patients with non-valvular atrial fibrillation (NVAF) on direct oral anticoagulants (DOACs) or warfarin. METHODS: MEDLINE, Embase, NHS-EED, and EconLit were searched for RWD studies published between January 2003 and November 2016 comparing MB risk among DOACs and warfarin. Proceedings of clinical conferences from 2012 to 2016 were reviewed. RESULTS: A total of 4218 citations were identified, 26 of which met eligibility criteria. Most studies were retrospective analyses of administrative claims databases and patient registries (n = 23 of 26); about half were based in the United States (n = 15). Apixaban showed a significantly lower risk of MB versus warfarin in all eight included studies. MB risk was either significantly lower (n = 9 of 16) or not significantly different (n = 7 of 16) between dabigatran and warfarin; there was no significant difference between rivaroxaban and warfarin in all seven included studies. The risk was significantly lower with apixaban versus rivaroxaban (n = 7 of 7) but not significantly different from dabigatran (n = 6 of 7). MB risk was significantly lower (n = 3 of 4) or not significantly different (n = 1 of 4) with dabigatran versus rivaroxaban. No evidence was identified for edoxaban. CONCLUSION: DOACs were associated with similar or lower risks of MB versus warfarin. A lower MB risk was consistently observed for apixaban, but less consistently for dabigatran; MB risk was similar between rivaroxaban and warfarin. Among DOACs, the risk of MB with apixaban was consistently lower than with rivaroxaban, but similar to dabigatran.

Involvement of α2-adrenoceptors in inhibitory and facilitatory pain modulation processes.

BACKGROUND: In healthy humans, high-frequency electrical stimulation (HFS) of the forearm not only produces hyperalgesia at the site of stimulation but also reduces sensitivity to pressure-pain on the ipsilateral side of the forehead. In addition, HFS augments the ipsilateral trigeminal nociceptive blink reflex and intensifies the ipsilateral component of conditioned pain modulation. The aim of this study was to determine whether α2-adrenoceptors mediate these ipsilateral nociceptive influences. METHODS: The α2-adrenoceptor antagonist yohimbine was administered to 22 participants in a double-blind, placebo-controlled crossover study. In each session, thermal and mechanical sensitivity in the forearms and forehead was assessed before and after HFS. In addition, the combined effect of HFS and yohimbine on the nociceptive blink reflex and on conditioned pain modulation was explored. In this paradigm, the conditioning stimulus was cold pain in the ipsilateral or contralateral temple, and the test stimulus was electrically evoked pain in the forearm. RESULTS: Blood pressure and electrodermal activity increased for several hours after yohimbine administration, consistent with blockade of central α2-adrenoceptors. Yohimbine not only augmented the nociceptive blink reflex ipsilateral to HFS but also intensified the inhibitory influence of ipsilateral temple cooling on electrically evoked pain at the HFS-treated site in the forearm. Yohimbine had no consistent effect on primary or secondary hyperalgesia in the forearm or on pressure-pain in the ipsilateral forehead. CONCLUSIONS: These findings imply involvement of α2-adrenoceptors both in ipsilateral antinociceptive and pronociceptive pain modulation processes. However, a mechanism not involving α2-adrenoceptors appears to mediate analgesia in the ipsilateral forehead after HFS.

Inappropriate initial antibiotic treatment for complicated skin and soft tissue infections in hospitalized patients: incidence and associated factors.

We analyzed 525 hospitalized adults treated with intravenous antibiotic(s) for complicated skin and soft tissue infections (cSSTIs) to assess incidence of, and risk factors associated with, inappropriate initial antibiotic treatment (IIAT). IIAT was given to 22.5% of enrolled patients. The rate of IIAT did not vary by type of facility (academic versus community) but was significantly higher in rural than urban hospitals (38.9% versus 21.3%, P = 0.02). Pathogens were exclusively gram-positive in 68% of patients, exclusively gram-negative in 13%, and mixed in 19%. Staphylococcus aureus was the most frequently isolated pathogen (in 65%), 54% of which were methicillin-resistant. Significant independent risk factors for IIAT were: admission to a rural hospital (odds ratio = 2.34; 95% confidence interval: 1.06-5.19), dialysis treatment (3.86; 1.15-12.93), cancer other than non-melanoma skin cancer (5.23; 1.78-15.36), and infection with gram-negative (3.43; 1.79-6.60) or mixed (4.52; 2.62-7.78) pathogens. IIAT for cSSTIs was relatively frequent in these hospitalized patients, especially those with selected risk factors.

Economic outcomes of inappropriate initial antibiotic treatment for complicated skin and soft tissue infections: a multicenter prospective observational study.

This study examined economic outcomes associated with inappropriate initial antibiotic treatment (IIAT) in complicated skin and soft tissue infections using data from adults hospitalized and treated with intravenous antibiotic therapy. We specifically analyzed for the subsets of patients infected with methicillin-resistant Staphylococcus aureus (MRSA), with healthcare-associated (HCA) infections, or both. Data from 494 patients (HCA: 360; MRSA:175; MRSA + HCA: 129) showed the overall mean length of stay (LOS) was 7.4 days and 15.0% had the composite economic outcome of any subsequent hospital admissions, emergency department visits, or unscheduled visits related to the study infection. A total of 23.1% of patients had IIAT; after adjustments, these patients had longer LOS than patients without IIAT in the HCA cohort (marginal LOS = 1.39 days, P = 0.03) and the MRSA + HCA cohort (marginal LOS = 2.43 days, P = 0.01) and were significantly more likely to have the composite economic outcome in all study cohorts (odds ratio: overall = 1.79; HCA = 3.09; MRSA = 3.66; MRSA + HCA = 6.92; all P < 0.05).

Coexistence of ipsilateral pain-inhibitory and facilitatory processes after high-frequency electrical stimulation.

BACKGROUND: High-frequency electrical stimulation (HFS) of the human forearm evokes analgesia to blunt pressure in the ipsilateral forehead, consistent with descending ipsilateral inhibitory pain modulation. The aim of the current study was to further delineate pain modulation processes evoked by HFS by examining sensory changes in the arm and forehead; investigating the effects of HFS on nociceptive blink reflexes elicited by supraorbital electrical stimulation; and assessing effects of counter-irritation (electrically evoked pain at the HFS-conditioned site in the forearm) on nociceptive blink reflexes before and after HFS. METHODS: Before and after HFS conditioning, sensitivity to heat and to blunt and sharp stimuli was assessed at and adjacent to the conditioned site in the forearm and on each side of the forehead. Nociceptive blink reflexes were also assessed before and after HFS with and without counter-irritation of the forearm. RESULTS: HFS triggered secondary hyperalgesia in the forearm (a sign of central sensitization) and analgesia to blunt pressure in the ipsilateral forehead. Under most conditions, both HFS conditioning and counter-irritation of the forearm suppressed electrically evoked pain in the forehead, and the amplitude of the blink reflex to supraorbital stimuli decreased. Importantly, however, in the absence of forearm counter-irritation, HFS conditioning facilitated ipsilateral blink reflex amplitude to supraorbital stimuli delivered ipsilateral to the HFS-conditioned site. CONCLUSIONS: These findings suggest that HFS concurrently triggers hemilateral inhibitory and facilitatory influences on nociceptive processing over and above more general effects of counter-irritation. The inhibitory influence may help limit the spread of sensitization in central nociceptive pathways.

High frequency electrical stimulation concurrently induces central sensitization and ipsilateral inhibitory pain modulation.

BACKGROUND: In healthy humans, analgesia to blunt pressure develops in the ipsilateral forehead during various forms of limb pain. The aim of the current study was to determine whether this analgesic response is induced by ultraviolet B radiation (UVB), which evokes signs of peripheral sensitization, or by high-frequency electrical stimulation (HFS), which triggers signs of central sensitization. METHODS: Before and after HFS and UVB conditioning, sensitivity to heat and to blunt and sharp stimuli was assessed at and adjacent to the treated site in the forearm. In addition, sensitivity to blunt pressure was measured bilaterally in the forehead. The effect of ipsilateral versus contralateral temple cooling on electrically evoked pain in the forearm was then examined, to determine whether HFS or UVB conditioning altered inhibitory pain modulation. RESULTS: UVB conditioning triggered signs of peripheral sensitization, whereas HFS conditioning triggered signs of central sensitization. Importantly, ipsilateral forehead analgesia developed after HFS but not UVB conditioning. In addition, decreases in electrically evoked pain at the HFS-treated site were greater during ipsilateral than contralateral temple cooling, whereas decreases at the UVB-treated site were similar during both procedures. CONCLUSIONS: HFS conditioning induced signs of central sensitization in the forearm and analgesia both in the ipsilateral forehead and the HFS-treated site. This ipsilateral analgesia was not due to peripheral sensitization or other non-specific effects, as it failed to develop after UVB conditioning. Thus, the supra-spinal mechanisms that evoke central sensitization might also trigger a hemilateral inhibitory pain modulation process. This inhibitory process could sharpen the boundaries of central sensitization or limit its spread.

Skin and soft tissue infections in hospitalised patients with diabetes: culture isolates and risk factors associated with mortality, length of stay and cost.

AIMS/HYPOTHESIS: Skin and soft tissue infections (SSTIs) cause substantial morbidity in persons with diabetes. There are few data on pathogens or risk factors associated with important outcomes in diabetic patients hospitalised with SSTIs. METHODS: Using a clinical research database from CareFusion, we identified 3,030 hospitalised diabetic patients with positive culture isolates and a diagnosis of SSTI in 97 US hospitals between 2003 and 2007. We classified the culture isolates and analysed their association with the anatomic location of infection, mortality, length of stay and hospital costs. RESULTS: The only culture isolate with a significantly increased prevalence was methicillin-resistant Staphylococcus aureus (MRSA); prevalence for infection of the foot was increased from 11.6 to 21.9% (p < 0.0001) and for non-foot locations from 14.0% to 24.6% (p = 0.006). Patients with non-foot (vs foot) infections were more severely ill at presentation and had higher mortality rates (2.2% vs 1.0%, p < 0.05). Significant independent risk factors associated with higher mortality rates included having a polymicrobial culture with Pseudomonas aeruginosa (OR 3.1), a monomicrobial culture with other gram-negatives (OR 8.9), greater illness severity (OR 1.9) and being transferred from another hospital (OR 5.1). These factors and need for major surgery were also independently associated with longer length of stay and higher costs. CONCLUSIONS/INTERPRETATION: Among diabetic patients hospitalised with SSTI from 2003 to 2007, only MRSA increased in prevalence. Patients with non-foot (vs foot) infections were more severely ill. Independent risk factors for increased mortality rates, length of stay and costs included more severe illness, transfer from another hospital and wound cultures with Pseudomonas or other gram-negatives.

The incremental costs of recommended therapy versus real world therapy in type 2 diabetes patients.

BACKGROUND: The goals of diabetes management have evolved over the past decade to become the attainment of near-normal glucose and cardiovascular risk factor levels. Improved metabolic control is achieved through optimized medication regimens, but costs specifically associated with such optimization have not been examined. OBJECTIVE: To estimate the incremental medication cost of providing optimal therapy to reach recommended goals versus actual therapy in patients with type 2 diabetes. METHODS: We randomly selected the charts of 601 type 2 diabetes patients receiving care from the outpatient clinics of Massachusetts General Hospital March 1, 1996-August 31, 1997 and abstracted clinical and medication data. We applied treatment algorithms based on 2004 clinical practice guidelines for hyperglycemia, hyperlipidemia, and hypertension to patients' current medication therapy to determine how current medication regimens could be improved to attain recommended treatment goals. Four clinicians and three pharmacists independently applied the algorithms and reached consensus on recommended therapies. Mean incremental medication costs, the cost differences between current and recommended therapies, per patient (expressed in 2004 dollars) were calculated with 95% bootstrap confidence intervals (CIs). RESULTS: Mean patient age was 65 years old, mean duration of diabetes was 7.7 years, 32% had ideal glucose control, 25% had ideal systolic blood pressure, and 24% had ideal low-density lipoprotein cholesterol. Care for these diabetes patients was similar to that observed in recent national studies. If treatment algorithm recommendations were applied, the average annual medication cost/patient would increase from 1525 to 2164 dollars. Annual incremental costs/patient increased by 168 dollars (95% CI 133-206 dollars) for antihyperglycemic medications, 75 dollars (57-93 dollars) for antihypertensive medications, 392 dollars (354-434 dollars) for antihyperlipidemic medications, and 3 dollars (3-4 dollars) for aspirin prophylaxis. Yearly incremental cost of recommended laboratory testing ranged from 77-189 dollars/patient. LIMITATIONS: Although baseline data come from the clinics of a single academic institution, collected in 1997, the care of these diabetes patients was remarkably similar to care recently observed nationally. In addition, the data are dependent on the medical record and may not accurately reflect patients' actual experiences. CONCLUSION: Average yearly incremental cost of optimizing drug regimens to achieve recommended treatment goals for type 2 diabetes was approximately 600 dollars/patient. These results provide valuable input for assessing the cost-effectiveness of improving comprehensive diabetes care.

The effect of rabeprazole on regional gastric acidity and the postprandial cardia/gastro-oesophageal junction acid layer in normal subjects: a randomized, double-blind, placebo-controlled study.

BACKGROUND: Postprandial intragastric acidity is not uniform. Postprandial proximal gastric acid pockets have been described in the present study. AIM: To determine the effects of rabeprazole on regional intragastric acidity and proximal acid pockets. METHODS: Ten normal subjects underwent two 8-day oral dosing regimens with placebo or rabeprazole 20 mg each morning in a randomized, double-blind protocol. Oesophago-gastric pH monitoring was performed on days 1 and 8. RESULTS: Rabeprazole increased fasting and postprandial gastric pH to above 4 in each area of the stomach on days 1 and 8. With placebo, acid pockets were identified at the cardia/gastro-oesophageal junction in 62 and 50 of 150 pull-throughs on days 1 and 8, respectively. Acid pockets were detected postprandially 3.1 +/- 0.2-5.8 +/- 0.1 cm below the proximal border of the lower oesophageal sphincter with a mean pH drop from 4.6 +/- 0.1 to 1.5 +/- 0.1. Rabeprazole decreased the number of acid pockets to 30 and 27 on days 1 and 8, respectively. Rabeprazole also decreased their length and magnitude of the pH drop. CONCLUSIONS: Rabeprazole increased intragastric pH on day 1 and 8 and maintained an elevated pH during and after meals. Postprandial acid pockets, identified in the region of the cardia/gastro-oesophageal junction area postprandially, were decreased in number, length and magnitude by rabeprazole.

Mpe1, a zinc knuckle protein, is an essential component of yeast cleavage and polyadenylation factor required for the cleavage and polyadenylation of mRNA.

In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene, MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice of ACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3'-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3' end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3' end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.

Lowering of blood glucose to nondiabetic levels in a hyperglycemic pig by allografting of fetal pig isletlike cell clusters.

BACKGROUND: Fetal pig isletlike cell clusters (ICCs) will differentiate when grafted into the thymus gland of outbred immunosuppressed nondiabetic pigs for up to 3 months. Whether these cells will survive for a similar period in a diabetic recipient and will mature with secretion of insulin to ameliorate the hyperglycemia is unknown. METHODS: Between 40,000 and 125,000 ICCs (7,000 to 11,400 ICCs/kg) were injected into the thymus gland of five juvenile pigs immunosuppressed with cyclosporine and deoxyspergualin, and the animals were subsequently made diabetic by the injection of streptozotocin. Insulin was administered subcutaneously, with one pig dying from hypoglycemia. The animal with the least number of ICCs transplanted was killed 81 days later, and the graft was analyzed histologically. Blood glucose levels and porcine C-peptide in the remaining animals were monitored for a median of 101 days. RESULTS: Histological analysis of the graft showed numerous epithelial cell clusters; the percentage of cells that contained insulin, glucagon, somatostatin, and pancreatic polypeptide were 61%, 64%, 25%, and 18%, respectively. Some cells contained more than one hormone. Porcine C-peptide was detected from 21 days after induction of diabetes but not before. In the pig receiving the most ICCs, blood glucose levels were lowered to nondiabetic levels 109 days after transplantation. Plasma C-peptide levels in response to glucagon in this pig steadily increased after grafting; peak levels were 0, 0.21, 0.45, and 0.52 ng/ml at 4, 21, 49, and 80 days after induction of diabetes compared to 0.09 ng/ml in control diabetic pigs. The secretion of C-peptide in response to oral and intravenous glucose and arginine also was greater than in untransplanted diabetic pigs, the pattern of secretion being consistent with developing fetal beta cells as the source of the C-peptide. Pancreatic insulin content was 0.1 mU/mg, 4% of that in nondiabetic pigs, and the number of beta cells per islet was 3 to 6 compared to 90 in nondiabetic controls. CONCLUSIONS: ICCs will differentiate and function for up to 111 days when transplanted into outbred immunosuppressed pigs rendered diabetic. Blood glucose levels can be lowered to nondiabetic levels when sufficient numbers of ICCs are grafted.

Autofluorescence of skin burns detected by fiber-optic confocal imaging: evidence that cool water treatment limits progressive thermal damage in anesthetized hairless mice.

BACKGROUND: Although full-thickness burns present no difficulty to clinical judgment, accurate assessment of burn depth immediately after injury in partial thickness burns has always been difficult. METHODS: Thermal burns (applied by a 3-mm-diameter brass rod heated to 50 degrees--80 degrees C for 20 seconds) were induced on the skin of anesthetized hairless mice. Anesthesia was maintained throughout all experiments. Both burns and normal skin were investigated noninvasively in vivo using fiber-optic confocal imaging (FOCI) microscopy (excitation, 488 nm; detection, 505 nm). RESULTS: Autofluorescence was detected in burned skin, and the depth of the autofluorescent region was found to correlate with the intensity of heat applied. Cool water treatment (for 20 minutes immediately after burn induction) significantly reduced the progressive increase in autofluorescence in deeper layers of the skin over the 4-hour postburn observation period. Histology showed burn-associated changes at a lower temperature than that at which autofluorescence was first detected in vivo by FOCI. However, there was a good correlation (r = 0.78) between depth of damage revealed by FOCI compared with that by histology. CONCLUSION: These results suggest that FOCI may be used to provide an index of burn depth.

In vivo detection of small subsurface melanomas in athymic mice using noninvasive fiber optic confocal imaging.

Fiber optic confocal imaging, following intravenous administration of fluorescently labeled antibodies and Texas Red-dextran, enabled in vivo detection of melanoma and surrounding blood vessels in athymic mice. Human melanoma cells (three cell lines) and cultured normal human skin cells were implanted intradermally into the haunch skin of anesthetized athymic BALB/C mice and allowed to grow to a maximum size of 2 mm diameter. Using three different fluorescein-isothiocyanate-labeled antimelanoma antibodies, single channel confocal images of melanoma cells were obtained in vivo. Using noninvasive techniques, the overall in vivo melanoma detection rate for tumors within 0.2 mm of the skin surface was 84% (27 of 32 tumors). Normal cultured human skin cells were found to have little or no fluorescence after administration of the fluorescein-isothiocyanate-labeled antibodies and tumors were not labeled by an isotype control antibody. Dual channel imaging of the implanted melanoma tumor and surrounding dermal vasculature in vivo showed increased blood vessel density at the melanoma site. Conventional immunoperoxidase histology confirmed that fiber optic confocal imaging was able to detect melanoma tumors up to 0.2 mm below the skin surface, in vivo.

Titanium(IV) isopropoxide mediated solution phase reductive amination on an automated platform: application in the generation of urea and amide libraries.

Amine libraries and their derivatives are important targets for high throughput synthesis because of their versatility as medicinal agents and agrochemicals. As a part of our efforts towards automated chemical library synthesis, a titanium(IV) isopropoxide mediated solution phase reductive amination protocol was successfully translated to automation on the Trident(TM) library synthesizer of Argonaut Technologies. An array of 24 secondary amines was prepared in high yield and purity from 4 primary amines and 6 carbonyl compounds. These secondary amines were further utilized in a split synthesis to generate libraries of ureas, amides and sulfonamides in solution phase on the Trident(TM). The automated runs included 192 reactions to synthesize 96 ureas in duplicate and 96 reactions to synthesize 48 amides and 48 sulfonamides. A number of polymer-assisted solution phase protocols were employed for parallel work-up and purification of the products in each step.

Deletion of the PAT1 gene affects translation initiation and suppresses a PAB1 gene deletion in yeast.

The yeast poly(A) binding protein Pab1p mediates the interactions between the 5' cap structure and the 3' poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. The PAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.

In vivo mapping of the vascular changes in skin burns of anaesthetised mice by fibre optic confocal imaging (FOCI).

Burns (3 mm in diameter, 50 degrees C, 20 s duration) were induced on the skin of anaesthetised hairless mice. Anaesthesia was maintained throughout all experiments. Subsurface changes in the microvasculature at the burn site were imaged confocally following i. v. injection of fluorescently labelled (FITC) dextran. Blood cells moving through dermal blood vessels were seen and recorded on video tape. Multiple adjacent 2-D confocal images of the burn site and surrounding areas were assembled and enabled microscopic vascular imaging of the whole burn area (including zones of coagulation, stasis and hyperaemia) and the surrounding normal vessels. This mapping of the burn area by fibre optic confocal imaging (FOCI) in vivo demonstrated good congruence with vascular casts (Microfil MV-120, Flow tech, USA) made at 4 h post burn. This study demonstrates the usefulness of FOCI for in vivo vascular imaging in burns.

Independent and hetero-oligomeric-dependent sperm binding to egg envelope glycoprotein ZPC in Xenopus laevis.

Vitelline envelopes are composed of glycoproteins that participate in sperm-egg interactions during the initial stages of fertilization. In Xenopus laevis, the vitelline envelope is composed of at least 4 glycoproteins (ZPA, ZPB, ZPC, and ZPX). A sperm binding assay involving the covalent coupling of envelope glycoproteins to silanized glass slides was developed. In our assay, sperm bound to the egg envelopes derived from oviposited eggs but not activated eggs. The majority of the egg envelope ligand activity for sperm binding was derived from the complex N-linked oligosaccharides of ZPC. This sperm binding involved N-acetylglucosamine and fucose residues, as binding was abolished after treatment with cortical granule beta-N-acetylglucosaminidase and commercial beta-N-acetylglucosaminidases and was reduced by 44% after treatment with alpha-fucosidase. Although both the envelope glycoproteins ZPA and ZPC possessed independent ligand activity, ZPC was the major ligand for sperm binding (75%). Mixing of isolated ZPA, ZPB, and ZPC in a ratio of 1:4:4 (equal to that in the egg envelope) resulted in sperm binding that was greater than that of the sum of the separate components. The egg glycoproteins acted in synergy to increase sperm binding. Thus, ZPC possessed both independent and hetero-oligomeric-dependent ligand activities for sperm binding.