Low On-Treatment Platelet Reactivity Predicts Long-Term Risk of Bleeding After Elective PCI.

BACKGROUND: Bleeding after percutaneous coronary interventions (PCI) is an important complication with impact on prognosis. AIM: To evaluate the predictive value of enhanced platelet responsiveness to dual antiplatelet therapy with aspirin and clopidogrel, for bleeding, after elective PCI. METHODS AND RESULTS: We performed multiple electrode aggregometry (MAE) platelet functional tests induced by arachidonic acid (ASPI) and adenosine-diphosphate (ADP) before PCI, and 24 hours after PCI, in 481 elective PCI patients who were followed-up for an average of 15.34 ± 7.19 months. Primary end point was the occurrence of any bleeding, while ischemic major adverse cardiovascular event (MACE) was a secondary endpoint. The incidence of total, BARC ≤ 2, and BARC ≥ 3 bleeding, according to BARC classification, was 19, 18, and 1%, respectively. Groups with any, and BARC ≤ 2 bleeding, had a lower average value of MAE ADP test after 24 hours, compared to the group without bleeding: 45.30 ± 18.63 U versus 50.99 ± 19.01 U; P = 0.005; and 45.75 ± 18.96 U versus 50.99 ± 18.99 U; P = 0.01; respectively. Female gender (HR 2.11; CI 1.37-3.25; P = 0.001), previous myocardial infarction (HR 0.56; CI 0.37-0.85; P = 0.006), lower body mass (HR 0.78; CI 0.62-0.98; P = 0.03), and MAE ADP test after 24 hours (HR 0.75; CI 0.61-0.93; P = 0.009) were the independent predictors for any bleeding by Cox univariate analysis. After adjustment, MAE ADP test after 24 hours, was the only independent predictor for any (HR 0.7; CI 0.56-0.87; P = 0.002), and BARC ≤ 2 (HR 0.71; CI 0.56-0.89; P = 0.003) bleeding, by Cox multivariate analysis. CONCLUSION: MAE ADP test before and after PCI, was associated with any, and BARC ≤ 2 bleeding after elective PCI.

International multicenter tool to predict the risk of four or more tumor-positive axillary lymph nodes in breast cancer patients with sentinel node macrometastases.

Recently, many centers have omitted routine axillary lymph node dissection (ALND) after metastatic sentinel node biopsy in breast cancer due to a growing body of literature. However, existing guidelines of adjuvant treatment planning are strongly based on axillary nodal stage. In this study, we aim to develop a novel international multicenter predictive tool to estimate a patient-specific risk of having four or more tumor-positive axillary lymph nodes (ALN) in patients with macrometastatic sentinel node(s) (SN). A series of 675 patients with macrometastatic SN and completion ALND from five European centers were analyzed by logistic regression analysis. A multivariate predictive model was created and validated internally by 367 additional patients and then externally by 760 additional patients from eight different centers. All statistical tests were two-sided. Prevalence of four or more tumor-positive ALN in each center's series (P = 0.010), number of metastatic SNs (P < 0.0001), number of negative SNs (P = 0.003), histological size of the primary tumor (P = 0.020), and extra-capsular extension of SN metastasis (P < 0.0001) were included in the predictive model. The model's area under the receiver operating characteristics curve was 0.766 in the internal validation and 0.774 in external validation. Our novel international multicenter-based predictive tool reliably estimates the risk of four or more axillary metastases after identifying macrometastatic SN(s) in breast cancer. Our tool performs well in internal and external validation, but needs to be further validated in each center before application to clinical use.

Metal-Phenolic Film Coated Quartz Crystal Microbalance as a Selective Sensor for Methanol Detection in Alcoholic Beverages.

The facile real-time monitoring of methyl content in fermented beverages is of fundamental significance in the alcohol and restaurant industry, since as little as 4 mL of methanol entering the blood may cause intoxication or blindness. So far, the practical applicability of available methanol sensors, including the piezoresonance analogs, is somewhat limited to laboratory use due to the complexity and bulkiness of the measuring equipment involving multistep procedures. This article introduces a hydrophobic metal-phenolic film-coated quartz crystal microbalance (MPF-QCM) as a novel streamlined detector of methanol in alcoholic drinks. Unlike other QCM-based alcohol sensors, our device operates under saturated vapor pressure conditions, permitting rapid detection of methyl fractions up to seven times below the tolerable levels in spirits (e.g., whisky) while effectively suppressing the cross-sensitivity to interfering chemical compounds such as water, petroleum ether or ammonium hydroxide. Furthermore, the good surface adhesion of metal-phenolic complexes endows the MPF-QCM with superior long-term stability, contributing to the repeatable and reversible physical sorption of the target analytes. These features, combined with the lack of mass flow controllers, valves and connecting pipes delivering the gas mixture, outline the likelihood for future design of a portable MPF-QCM prototype suitable to point-of-use analysis in drinking establishments.

Multidisciplinary management of acute cholecystitis during the COVID-19 pandemic.

The coronavirus disease 2019 pandemic had a major impact on most medical services. Our aim was to assess the outcome of acute cholecystitis during the nationwide lockdown period. All patients admitted to our emergency department for AC were analysed. Patient characteristics, performance status, AC severity, treatment modality and outcome of AC were assessed during the lockdown period (Period II: 1 April 2020-30 November 2021) and compared to a historical control period (Period I: 1 May 2017-31 December 2018). AC admissions increased by 72.8% in Period II. Patients were younger (70 vs. 74 years, p = 0.017) and greater in number in the CCI 1 group (20.4% vs. 11.2%, p = 0.043) in Period II. The unplanned readmission rate (6.3 vs. 0%, p = 0.004) and the gallbladder perforation (GP) rate was higher (18.0 vs. 7.3%, p = 0.006) in Period II. Percutaneous transhepatic gallbladder drainage (PTGBD) was more frequent (24.1 vs. 12.8%, p = 0.012) in Period II. In addition to a drop in patient age and CCI, a significant rise in the prevalence of acute cholecystitis, GP and unplanned readmissions was observed during the nationwide lockdown due to the COVID-19 pandemic. PTGBD was more frequent during this period, whereas successful conservative treatment was less frequent.

The relationship of multifocality and tumor burden with various tumor characteristics and survival in early breast cancer.

The presence of multifocality and the aggregate tumor size were retrospectively analysed in a database of 1071 operated breast cancers. Around a quarter of all these cancers involved multiple foci, while a tenth of the total demonstrated more than one invasive focus. Although the multifocal cancers were smaller and more often screen-detected than the unifocal cancers, their aggregate tumor size was larger, and they more frequently displayed casting-type calcifications in the mammogram and HER2 positivity. Lobular histology favoured larger tumor burden. The invasive multifocal cancers were more commonly lymph node-positive than the other tumors. In a subgroup of 584 patients with a median follow-up time of 5 years, the larger size of the invasive tumor, the presence of LVI or lymph node involvement, HER2 positivity and triple negativity were associated with a poorer RFS and OS, while the outcome of screen-detected tumors was superior to that of non-screen-detected or interval cancers. A large tumor size, lymph node positivity and HER2 positive or triple negative phenotypes were independent determinants of a poorer survival rate.

Detailed esophageal function and morphological analysis shows high prevalence of gastroesophageal reflux disease and Barrett's esophagus in patients with cervical inlet patch.

Although the pathogenesis of cervical inlet patch (CIP) is not fully understood, most authors consider it as a congenital abnormality, whereas others surmise it to be related to gastroesophageal reflux disease (GERD). We aimed to evaluate esophageal function and the prevalence of GERD and Barrett's esophagus in patients with CIP. GERD is defined by the presence of erosive esophagitis or an abnormal pH monitoring. Seventy-one consecutive patients with endoscopic and histological evidence of CIP were prospectively evaluated. Esophageal symptom analysis, 24-hour simultaneous biliary reflux and double-channel pH-monitoring, and esophageal manometry were carried out in 65/71 (92%) patients and in 25 matched controls. Six patients were not suitable for testing and were, therefore, excluded. The histological evaluation of the heterotopic islands showed cardia and/or oxyntic mucosa in 64/65 (98%) patients and specialized intestinal metaplasia (SIM) in one patient (2%). The cardia and/or oxyntic mucosa was accompanied by focally appearing pancreatic acinar metaplasia and pancreatic ductal metaplasia in 7/64 (11%) and in 1/64 (2%), superficial mucous glands in 6/64 (9%), and SIM in 2/64 (3%) cases. In total, SIM was present in three patients (5%), and one of them had low-grade dysplasia. At the gastroesophageal junction, 28 (43%) patients had columnar metaplasia, including nine (14%) patients with SIM. Erosive esophagitis was present in 37 (57%) cases. Thirty-two patients (49%) had abnormal acid reflux in the distal and 25 (38%) in the proximal esophagus. Abnormal biliary reflux was present in 25 (38%) cases. On the basis of endoscopic and pH studies, GERD was established in 44/65 (68%) patients. Typical reflux symptoms were common (33/65, 51%). The combined 24-hour biliary and double-channel pH-monitoring detected significantly more significant acidic reflux at both measurement points and significantly longer bile exposure time in the distal esophagus in patients with CIP. Acid secretion in the CIP was detected in three (5%) cases. Esophageal manometry revealed decreased LES pressure and prolonged relaxation with decreased peristaltic wave amplitude, and an increased number of simultaneous contractions in the esophageal body. The detailed evaluation of the esophageal morphology and function in subjects with CIP showed a high prevalence of GERD and Barrett's esophagus. Further studies are needed to evaluate whether combined acidic and biliary reflux is able to promote similar histomorphological changes in the CIP, as it is shown distally in patients with Barrett's esophagus.

Is axillary lymph node dissection necessary for positive preoperative aspiration cytology lymph node results?

INTRODUCTION: Based on international guidelines, axillary lymph node dissection (ALND) is recommended in cases of breast cancer if preoperative examinations confirm axillary metastasis. We examined which set of preoperative parameters might render ALND unnecessary. PATIENTS AND METHODS: Preoperative examinations (axillary ultrasound and aspiration cytology) confirmed axillary metastasis in 190 cases out of 2671 patients with breast cancer; primary ALN dissection was performed on these patients with or without prior neoadjuvant therapy. The clinicopathological results were analysed to determine which parameter might predict the presence of no more than 2 or 3 metastatic ALNs. RESULTS: The final histological examination confirmed 1-3 metastatic lymph nodes in ALND samples in 116 cases and over 3 metastatic lymph nodes in 74 cases. For patients receiving neoadjuvant therapy (59 out of the 190 cases), if the size of the primary tumour was 2 cm or smaller and/or the metastatic ALN was 15 mm or smaller, then the patient was likely to have no more than 3 positive ALNs (stage N0-1 disease) (p < 0.001). If the patient did not receive neoadjuvant therapy, stage N2 or N3 disease was very likely. No correlation was found between other clinicopathological characteristics of the tumour and involvement of the ALNs. CONCLUSION: Axillary lymph node dissection is not necessary for selected breast cancer patients with axillary metastasis receiving neoadjuvant therapy. In these cases, sentinel lymph node biopsy with or without radiation therapy and close follow-up may serve as adequate therapy.

Early microcirculatory changes after ischemic preconditioning and small bowel autotransplantation.

BACKGROUND/AIMS: Ischemia-reperfusion injury contributes to the high complication rate of small bowel transplantation (SBTX). Ischemic preconditioning (IPC) protects against reperfusion injury in several organs, but the IPC-induced microcirculatory reaction in the intestine is unknown. METHODS: We examined the effects of IPC on the macrohemodynamics and graft microcirculation in a canine model of SBTX during a 4-hour reperfusion period. In group 1 SBTX was performed, in group 2 IPC was induced before graft harvesting (ischemia 3 times for 5 min, followed by 10 min of reperfusion). Cardiac index and mesenteric blood flow were measured, and the mucosal microcirculation, villus epithelial thickness and functional capillary density were monitored by orthogonal polarization spectral imaging. Leukocyte-endothelial cell interactions were monitored in the postcapillary venules, with intravital fluorescence microscopy. RESULTS: Reperfusion decreased cardiac index and mesenteric blood flow during reperfusion; IPC significantly improved these changes. Reperfusion was accompanied by decreased functional capillary density and epithelial thickness of the villi and increased leukocyte-endothelial cell interactions. IPC increased functional capillary density, prevented epithelial narrowing and reduced leukocyte rolling and adherence. CONCLUSION: IPC improves the macrohemodynamics and the intestinal microcirculation and reduces leukocyte-mediated tissue injury during reperfusion. IPC can be an effective tool to limit reperfusion injury during SBTX.

Post-transplantation morphological and functional changes in kidneys from expanded criteria donors.

Introduction Despite an increase in the number of cadaver donors and overall organ transplantations, the dramatic increase in the waiting list makes it necessary to reconsider donor criteria. The authors wanted to examine whether differences could exist in the function and/or morphology of transplanted kidneys originated from expanded criteria donors (ECDs) and ideal donors 1 and 5 years after transplantation. Methods Kidney function and histopathologic findings were analyzed and compared 1 and 5 years after transplantation in 97 patients having ECD kidneys and in 178 patients who received ideal donor kidneys (IDK). Results Serum creatinine level was significantly higher (p = 0.001) and estimated glomerular filtration rate was significantly lower (p = 0.003) in patients having ECD kidneys as compared with those with IDK 5 years after transplantation. Morphological changes in the transplanted kidneys, such as tubulitis (p = 0.025) and interstitial inflammation (p = 0.002), were significantly more frequently present in patients with ECD kidneys than in those with IDK 1 year after transplantation. Conclusion Despite an absence of differences in kidney function 1 year after kidney transplantation between patients having ECD and IDK, morphological differences in the transplanted kidneys can be detected between the two groups of patients.

Is intraoperative touch imprint cytology indicated in the surgical treatment of early breast cancers?

INTRODUCTION: Intraoperative touch imprint cytology (TIC) of the sentinel lymph node(s) (SLN(s)) in the treatment of breast cancer has significantly reduced the number of axillary block dissections (ABD) required during second surgeries. Based on recent studies, ABD was not considered necessary if the presence of tumor cells/micrometastasis was confirmed in the SLN(s) or in the case of macrometastases in a patient group meeting the inclusion criteria for the ACOSOG Z0011 study. Our aim was to determine the sensitivity and usefulness of TIC with regard to these results. METHODS: TICs of the SLN(s) were examined in 1168 patients operated on for breast cancer. The method was also analyzed retrospectively based on the guidelines for the Z0011 study. During TIC, new samples were cut every 250 µm; impression smears were evaluated after being stained with hematoxylin eosin. RESULTS: TIC confirmed metastasis in 202 cases (202/1168, 17.29%). Metastasis was confirmed in SLN(s) in 149 additional cases during a final histological examination. The sensitivity of TIC was found to be 57.18%, and its specificity was 99.63%. An analysis was then performed except for cases that met the inclusion criteria for the Z0011 study and with metastasis smaller than 2 mm (micrometastasis/isolated tumor cells) considered to be positive during intraoperative cytology. The sensitivity of the method decreased to 34.23%, while its specificity was still high at 99.76%. CONCLUSIONS: Based on the new guidelines for ABD, imprint cytology cannot be considered a beneficial and cost-effective intervention in the surgical treatment of early breast cancer.

Solution structure and dynamics of a designed hydrophobic core variant of ubiquitin.

BACKGROUND: The recent merger of computation and protein design has resulted in a burst of success in the generation of novel proteins with native-like properties. A critical component of this coupling between theory and experiment is a detailed analysis of the structures and stabilities of designed proteins to assess and improve the accuracy of design algorithms. RESULTS: Here we report the solution structure of a hydrophobic core variant of ubiquitin, referred to as 1D7, which was designed with the core-repacking algorithm ROC. As a measure of conformational specificity, we also present amide exchange protection factors and backbone and sidechain dynamics. The results indicate that 1D7 is similar to wild-type (WT) ubiquitin in backbone structure and degree of conformational specificity. We also observe a good correlation between experimentally determined sidechain structures and those predicted by ROC. However, evaluation of the core sidechain conformations indicates that, in general, 1D7 has more sidechains in less statistically favorable conformations than WT. CONCLUSIONS: Our results provide an explanation for the lower stability of 1D7 compared to WT, and suggest modifications to design algorithms that may improve the accuracy with which structure and stability are predicted. The results also demonstrate that core packing can affect conformational flexibility in subtle ways that are likely to be important for the design of function and protein-ligand interactions.

Gadolinium chloride-induced shifts in intrahepatic distributions of liposomes.

Intravenously administered gadolinium chloride caused only a slight decrease in the rate of elimination of small unilamellar liposomes from the blood and had no influence on the total hepatic uptake of these vesicles, but did alter their intrahepatic distribution substantially. Uptake by the non-parenchymal cells was substantially decreased, whereas uptake by the parenchymal cells showed a concomitant increase. Our earlier observations (Roerdink et al. (1981) Biochim. Biophys. Acta 677, 79-89) on the effect of lanthanides on the in vivo distribution of multilamellar liposomes have been extended, in that we demonstrate, in addition to the drop in elimination rate from the blood and in the over-all hepatic uptake, a shift of liposome distribution within the Kupffer cell population. While the larger Kupffer cells, which normally take up a major fraction of an injected liposome dose, were strongly inhibited in liposome uptake, the more numerous small macrophages showed a 3-4-fold increase in uptake.

Purification and characterization of the activated mineralocorticoid receptor from rat myocardium.

Cardiac cytosol from adrenalectomized rats was radiolabelled with 10 nM tritiated RU 26752, R 5020 or aldosterone, to saturate the mineralocorticoid receptor (MCR) in the presence of 1 microM RU 38486 to block the glucocorticoid and progestin receptors. Free steroids were removed by charcoal treatment and the radiolabelled cytosol was passed through a phosphocellulose column. The MCR peak in the phosphocellulose eluate was activated at 25 degrees C for 45 min, adsorbed onto the DNA-cellulose and finally extracted once each with buffers containing 1 M potassium chloride or 25 mM magnesium chloride. The pooled DNA-cellulose extracts, reequilibrated with 10 nM [3H]RU 26752, were resolved as a single, homogeneous band of 78 kDa upon polyacrylamide gel electrophoresis. Ion-exchange analysis of the purified MCR on DEAE-cellulose-52 revealed a single peak in the 0.017 M sodium phosphate region with both RU 26752 and R 5020, but aldosterone dissociated during this procedure. Molecular filtration on Ultrogel AcA-44 columns revealed a major 145 kDa peak, with some smaller components of 40 and 80 kDa. These hydrodynamic properties of the purified MCR are at variance with those of the native receptor in crude myocardial cytosol, and suggest that some post-translational modifications in vivo may be required for the expression of MCR-mediated responses.

Hydrophobic core packing and protein design.

Over the past few years, we have witnessed exciting advances in protein design. Several groups have reported success in the design of hydrophobic cores, and the principles developed in these studies have been recently applied to the full sequence design of a small protein motif and the design of a catalytically active metal center. These successes suggest that designing large, functional proteins in computero is more feasible than ever before.

Expression of cycloheximide resistance in carrot somatic hybrids and their segregants.

Cycloheximide resistance (CH(r)) was shown to be a function expressed in differentiated plant tissues, but not in unorganized callus tissues. A variant, WCH105, expressing CH(r) in the callus, as well as in regenerated plantlets, was isolated from a cell line derived from a wild carrot plant. The plantlets regenerated from WCH105 are green, but do not produce normal, dissected leaves. Protoplasts of WCH105 were fused with that of a cycloheximidesensitive (CH(s)) cell line derived from an albino, domesticated carrot. Hybrid selection was based on (1) irreversible growth inhibition of WCH105 protoplasts by iodacetamide, and (2) restoration of green plants producing dissected leaves.--Analysis of the CH(r) trait as an unselected marker in the callus cells of the somatic hybrids indicated that it behaved as a recessive. The combined recessive and resistant phenotype of this trait allowed the recovery of CH(r) segregants from CH(s) hybrids at a frequency of 10(-4), 1000 times higher than the spontaneous frequency of CH(r). The recovery of CH(r) somatic segregants confirmed the recessiveness of the CH(r) trait.

Distal renal tubular acidosis in multiple myeloma.

A patient with early multiple myeloma was initially seen with a severe hyperchloremic metabolic acidosis with a normal anion gap and a urine pH of 6.3. The patient did not have glucosuria, aminoaciduria, of phosphaturia. A bicarbonate loading test showed that the fractional excretion of bicarbonate was less than 5% and confirmed the hypothesis that the patient had a distal renal tubular acidification defect. The pathophysiologic mechanism that caused this defect is unknown, but it is associated with the presence of a serum M component (IgG-lambda) and a urine M component (lambda light chains). Multiple myeloma should be considered in the differential diagnosis of conditions of patients who have a renal tubular acidification defect.

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.

Rotamer strain as a determinant of protein structural specificity.

We present direct evidence for a change in protein structural specificity due to hydrophobic core packing. High resolution structural analysis of a designed core variant of ubiquitin reveals that the protein is in slow exchange between two conformations. Examination of side-chain rotamers indicates that this dynamic response and the lower stability of the protein are coupled to greater strain and mobility in the core. The results suggest that manipulating the level of side-chain strain may be one way of fine tuning the stability and specificity of proteins.

Effect of RU 38486 on TNF production and toxicity.

Glucocorticoid steroids provide considerable protection against the systemic toxicity of tumor necrosis factor-alpha (TNF-alpha, cachexin). In animal experiments RU 38486 (mifepristone), a steroid antagonist, increased the synthesis of TNF and sensitized the animals to the cytotoxic action of TNF. As compared to the control and methylprednisolone-treated groups, mifepristone significantly increased the level of TNF in the serum, liver and spleen of lipopolysaccharide (LPS)-treated animals. In tissue cultures RU 38486 induced the TNF synthesis of myeloid cells and increased the TNF production of genetically modified HeLa cells, which synthesize TNF constitutively. Normal and tumor cell cultures exhibited increased sensitivity toward TNF in the presence of mifepristone.

Cobalt injected into the right and left fasciculi retroflexes clarifies the organization of this pathway.

A cobaltic lysine complex was injected separately into the right and left fasciculi retroflexes of the frog. This tracing technique labeled, in a Golgi-like manner, the neurons which initiate the fasciculi retroflexes and revealed details of their morphological pattern. The fasciculi retroflexes originate from various neurons distributed in the diencephalon and mesencephalon, but their main source is the habenular nuclei. In the frog there are dorsal and ventral habenulae which are homologous to the medial and lateral habenulae, respectively, of mammals. In the frog the dorsal habenulae are strikingly asymmetric. Our study shows that the fasciculus retroflexus is composed of several compact separated bundles of fibers and that the fibers originating from the dorsal habenular nuclei project to the interpeduncular nucleus, while those originating from the ventral habenular nuclei project beyond the interpeduncular nucleus, but so far we have not been able to show the exact site of their termination. The labeling of cells within the interpeduncular nucleus raises the possibility that the habenulo-interpeduncular tract is reciprocal in function. The finds support our previously reported hypothesis on the theoretical interpretation of the functional circuitry of the frog habenulo-interpeduncular system.