Using the plant vacuole as a biological system to investigate the functional properties of exogenous channels and transporters.

Plant cells possess a large intracellular compartment that animal cells do not, the central vacuole, which has been investigated for a long time. The central vacuole can occupy up to 90% of the cellular volume and, differently from intracellular organelles from animal cells such as lysosomes or endosomes, it is easy to isolate. Because of its large dimension (up to 40 µm diameter) it can be successfully studied using the classical patch-clamp technique. Following the idea that the vacuolar membrane could be used as a convenient model to characterize the functional properties of channel-forming peptides, we verified that the phytotoxic lipodepsipeptide Syringopeptin 25A from Pseudomonas syringae pv syringae was able to form ionic pores in sugar beet vacuoles and we performed a detailed biophysical analysis. Recently, we extended the use of plant vacuoles to the expression and functional characterization of animal intracellular transporters, namely rat CLC-7, and channels, i.e. human TPC2. Since endo-lysosomal transporters and channels are still largely unexplored, principally because their intracellular localization renders them difficult to study, we believe that this novel approach will prove to be a powerful system for the investigation of the molecular mechanisms of exogenous transporters and channels. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

Pilot surveillance for childhood encephalitis in Australia using the Paediatric Active Enhanced Disease Surveillance (PAEDS) network.

We aimed to assess the performance of active surveillance for hospitalized childhood encephalitis in New South Wales (NSW) using the Paediatric Active Enhanced Disease Surveillance (PAEDS) network to inform methodology for the nationwide Australian childhood encephalitis (ACE) study. We piloted active surveillance for suspected encephalitis from May to December 2013 at the Children's Hospital at Westmead, Sydney, NSW. Cases were ascertained using four screening methods: weekday nurse screening of admission records (PAEDS), cerebrospinal fluid (CSF) microscopy records, magnetic resonance imaging (MRI) reports, and pharmacy dispensing records. Comprehensive clinical data were prospectively collected on consented participants and subsequently reviewed by an expert panel. Cases were categorized as confirmed encephalitis or 'not encephalitis'; encephalitis cases were sub-categorized as infectious, immune-mediated or unknown. We performed an ICD-10 diagnostic code audit of hospitalizations for the pilot period. We compared case ascertainment in the four screening methods and with the ICD code audit. Forty-eight cases of suspected encephalitis were identified by one or more methods. PAEDS was the most efficient mechanism (yield 34%), followed by MRI, CSF, and pharmacy audits (yield 14%, 12%, and 7% respectively). Twenty-five cases met the criteria for confirmed encephalitis. PAEDS was the most sensitive of the mechanisms for confirmed encephalitis (92%) with a positive predictive value (PPV) of 72%. The ICD audit was moderately sensitive (64%) but poorly specific (Sp 9%, PPV 14%). Of the 25 confirmed encephalitis cases, 19 (76%) were sub-categorized as infectious, three (12%) were immune-mediated, and three (12%) were 'unknown'. We identified encephalitis cases associated with two infectious disease outbreaks (enterovirus 71, parechovirus 3). PAEDS is an efficient, sensitive and accurate surveillance mechanism for detecting cases of childhood encephalitis including those associated with emerging infectious diseases. Active surveillance significantly increases the ascertainment of encephalitis cases compared with passive approaches.

Clinical Outcomes of Renin-Angiotensin-Aldosterone Blockade in Patients With Advanced Chronic Kidney Disease: A Systematic Review and Meta-analysis.

BACKGROUND: The cardiovascular and renal benefits of renin-angiotensin aldosterone system (RAAS) blockade are not well established in patients with advanced chronic kidney disease (CKD). We conducted a systematic review and meta-analysis to identify potential risks and benefits from RAAS blockade in patients with CKD stage 4-5. METHODS: A Medline search from inception to November 2022 was conducted to identify randomised controlled trials (RCTs) in patients with CKD stage 4-5 (estimated glomerular filtration rate ≤ 30 mL/min/1.73 m2) comparing RAAS blockade vs placebo or alternative antihypertensive therapy. Different intervention strategies were assessed (RAAS use vs nonuse, initiation vs placebo/alternative therapy, or discontinuation vs continuation). The primary outcome was progression to end-stage kidney disease (ESKD). Secondary outcomes were all-cause mortality and major adverse cardiovascular events (MACE). The risk ratio (RR) was estimated with the use of a random-effects model. RESULTS: Nine RCTs (1150 patients) were included. RAAS blockade was associated with a significant reduction in progression to ESKD: RR 0.84 (95% confidence interval [CI] 0.74-0.96; P = 0.01). There was no benefit from RAAS blockade on all-cause mortality or MACE: RR 1.02 (95% CI 0.63-1.65; P = 0.93) and RR 0.87 (95% CI 0.49-1.57; P = 0.65), respectively. CONCLUSIONS: RAAS blockade may be considered in selected patients with CKD stage 4-5 to delay progression to ESKD.

A perspective on the modulation of plant and animal two pore channels (TPCs) by the flavonoid naringenin.

The inhibitory effect of the flavonoid naringenin on plant and human Two-Pore Channels (TPCs) was assessed by means of electrophysiological measurements. By acting on human TPC2, naringenin, was able to dampen intracellular calcium responses to VEGF in cultured human endothelial cells and to impair angiogenic activity in VEGF-containing matrigel plugs implanted in mice. Molecular docking predicts selective binding sites for naringenin in the TPC structure, thus suggesting a specific interaction between the flavonoid and the channel.

Post-translational modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface.

Annexin A1 (ANXA1) has an important role in cell-cell communication in the host defense and neuroendocrine systems. In both systems, its actions are exerted extracellularly via membrane-bound receptors on adjacent sites after translocation of the protein from the cytoplasm to the cell surface of adjacent cells. This study used molecular, microscopic, and pharmacological approaches to explore the mechanisms underlying the cellular exportation of ANXA1 in TtT/GF (pituitary folliculo-stellate) cells. LPS caused serine-phosphorylation of ANXA1 (ANXA1-S27-PO4) and translocation of the phosphorylated protein to the cell membrane. The fundamental requirement of phosphorylation for membrane translocation was confirmed by immunofluorescence microscopy on cells transfected with wild-type or mutated (S27/A) ANXA1 constructs tagged with enhanced green fluorescence protein. The trafficking of ANXA1-S27-PO4 to the cell surface was dependent on PI3-kinase and MAP-kinase. It also required HMG-coenzyme A and myristoylation. The effects of HMG-coenzyme A blockade were overcome by mevalonic acid (the product of HMG-coenzyme A) and farnesyl-pyrophosphate but not by geranyl-geranylpyrophosphate or cholesterol. Together, these results suggest that serine-27 phosphorylation is essential for the translocation of ANXA1 across the cell membrane and also identify a role for isoprenyl lipids. Such lipids could target consensus sequences in ANXA1. Alternatively, they may target other proteins in the signal transduction cascade (e.g., transporters).

Annexin-1 downregulation in thyroid cancer correlates to the degree of tumor differentiation.

We investigated the expression of annexin-1 (ANXA1) in thyroid carcinoma cell lines and in thyroid cancers with a different degree of differentiation. The highest level of ANXA1 expression examined by Western blotting was detected in the papillary carcinoma cells (NPA) and in the follicular cells (WRO). On the other hand, the most undifferentiated thyroid carcinoma cells (ARO and FRO) presented the lowest level of ANXA1 expression. In surgical tissue specimens from 32 patients with thyroid cancers, we found high immunoreactivity for ANXA1 in papillary (PTC) and follicular (FTC) thyroid cancers while in undifferentiated thyroid cancers (UTC) the expression of the protein was barely detectable. Control thyroid tissue resulted positive for ANXA1. In summary, 70% of UTC examined weakly expressed ANXA1, whereas 65% of PTC or FTC specimens tested showed high expression of the protein. Thus ANXA1 expression may correlate with the tumorigenesis suggesting that the protein may represent an effective differentiation marker in thyroid cancer.

Scenedesmus dimorphus (Turpin) Kützing growth with digestate from biogas plant in outdoor bag photobioreactors.

Digestate coming from an Anaerobic Digestion unit in a Biogas Plant, feeded on cow manure and vegetable waste from markets, has been used. About 8-35 L polyethylene transparent bags have been employed as cultivation container, outdoor. Different aliquots of digestate, alone or mixed with commercial liquid fertiliser, were employed to cultivate in batch Scenedesus dimorphus, a freshwater green microalga, in the ENEA facilities of Casaccia Research Center, near Rome, Italy. The cultivation period was June-July 2013. The average daily yields of dry microalgae biomass varied from 20 mg/L/d to 60 mg/L/d, mean 38.2 mg/L/d. Final dry biomass concentration varied from 0.18 to 1.29 g/L, mean 0.55 g/L. S. dimorphus proved to be very efficient in removing N and P from the culture medium. Another fact emerged from these trials is that S. dimorphus inner composition resulted to be variable in response to the tested different culture conditions.

BAG3 regulates formation of the SNARE complex and insulin secretion.

Insulin release in response to glucose stimulation requires exocytosis of insulin-containing granules. Glucose stimulation of beta cells leads to focal adhesion kinase (FAK) phosphorylation, which acts on the Rho family proteins (Rho, Rac and Cdc42) that direct F-actin remodeling. This process requires docking and fusion of secretory vesicles to the release sites at the plasma membrane and is a complex mechanism that is mediated by SNAREs. This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the ß cell, hence facilitating insulin secretion. In this manuscript, we show for the first time that BAG3 plays an important role in this process. We show that BAG3 downregulation results in increased insulin secretion in response to glucose stimulation and in disruption of the F-actin network. Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1. Upon glucose stimulation BAG3 is phosphorylated by FAK and dissociates from SNAP-25 allowing the formation of the SNARE complex, destabilization of the F-actin network and insulin release.

Effect of zinc intake on copper excretion and retention in men.

To determine the influence of zinc intake on copper excretion and retention, nine men consumed diets containing 2.6 mg of copper/day and 1.8, 4.0, 6.0, 8.0, 18.5 or 20.7 mg of zinc/day for one- or two-week periods in a 63-day study. Copper and zinc in the diet and copper in plasma were determined weekly; fecal copper was determined daily and averaged within each week. The weekly mean (+/-SEM) plasma copper concentrations (81 +/- 3.3 to 100 +/- 5.8 micrograms/dl) remained within the normal range throughout the study. Fecal copper and apparent copper retention were influenced by the level of dietary zinc and the duration it was fed. When 18.5 mg of zinc/day was fed for two consecutive weeks following a lower zinc intake, fecal copper was elevated and apparent copper retention was reduced after a one-week lag. Thus, an intake of zinc only 3.5 mg/day above the RDA for men reduced apparent retention of copper at an intake of 2.6 mg/day.

Prosthetic valve obstruction: thrombolysis versus operation.

An acute obstruction is a life-threatening complication of mechanical valve prostheses, and is caused by the formation of fresh clot or fibrous tissue overgrowth, or both. Accurate selection of the most appropriate treatment for a particular patient is mandatory. From January 1991 to July 1992, 28 cases of prosthetic thrombosis were managed. Twenty patients underwent surgical treatment, with one operative death, and 8 patients were treated with thrombolysis using recombinant tissue-type plasminogen activator (rt-PA). The criteria for using thrombolysis were (1) the recent onset of symptoms, (2) transesophageal echocardiographic evidence of clots on the valve or cardiac chambers, and (3) preserved disc excursions. All patients who underwent thrombolysis had mechanical valves (two bileaflets, four tilting discs, and two ball valves); seven valves were in the mitral position and one was in the aortic. Symptoms of obstruction consisted of cardiac failure in 6 cases or thromboembolism in 5, or both. The mean interval between the onset of symptoms and the initiation of thrombolysis was 81 +/- 65 hours. After infusion of the rt-PA, normal valve function was restored in all patients, as documented by transesophageal echocardiography. No deaths or neurologic complications occurred; there was one episode of minor peripheral embolism. Thrombolysis using rt-PA may be the appropriate treatment in patients with primary thrombosis of mechanical valves, thereby avoiding the operation-related risks.

Prolapse of the "floppy" aortic valve as a cause of aortic regurgitation. A clinico-morphologic study.

A clinico-pathologic study was performed in 25 patients undergoing aortic valve replacement because of regurgitation, caused by myxoid degeneration of the valve leaflets. Associated cardiac anomalies were floppy mitral valve (2 cases), floppy mitral valve and idiopathic hypertrophic subaortic stenosis (1), left atrial myxoma (1), and aortic coarctation at the isthmus (1). Three patients died (2 immediately and 1 on the 30th postoperative day). Pathological studies of the explanted valves showed deformities characterized by redundant thin leaflets which appeared soft and gelatinous. On histologic examination the fibrous layer of the leaflets was seen to be infiltrated by myxomatous tissue. Echocardiography showed the aortic root to be dilated in 13 patients and normal in the others. In those with normal aortic root, the histological examination of aortic wall disclosed minimal cystic medial necrosis in two cases. In contrast, more severe forms of cystic medial necrosis were evident in all patients having a dilated aortic root. Aortic valve replacement was performed in all cases. It was accompanied by a Bentall procedure (1 case), repair of ascending aorta dissection (2), replacement of the ascending aorta (1), mitral valve replacement (2), mitral valve replacement and apico-ascending aorta conduit (1) and excision of a left atrial myxoma (1). Our experience suggests that prolapse of the aortic valve due to floppy leaflets is a common degenerative disease which is generally associated with noninflammatory aortic root degeneration. This, together with aortic root dilatation, contributes to valve insufficiency. Nevertheless, the disease, when isolated (with normal aortic root), is liable in itself to produce aortic regurgitation. The need for early diagnosis is stressed, so as to be able to perform valve replacement.

Clinical results after cardiac valve replacement with the Sorin prosthesis. A 6-year experience.

The mechanical valve produced by Sorin is a modified tilting disc device. Between March 1979 and November 1985, 763 Sorin prostheses were implanted in 710 patients. The study has been restricted to 504 isolated mitral or aortic valves. There were 238 females and 266 males whose ages ranged from 2 to 71 years (mean 43.8). Follow-up is 99.23% and covers 1045 patient-years. No structural failure has been observed. In the aortic position, 282 Sorin prostheses were implanted with the large orifice orientated towards the right coronary sinus. Hospital mortality was 5% (14/282). Actuarial survival at 78 months was 94.68% +/- 1.6% with a linearized mortality rate of 1.7% patient-years. There were 11 late deaths (9 valve-related and 2 non valve-related). The linearized rate of occurrence of embolic events was 0.32 patient-years (2/268). Actuarial survival complication-free at 78 months was 90.88% +/- 2.83%. Preoperatively, 19.8% patients were in NYHA class II, 68.5% in III and 11.7% in IV; postoperatively, 79.3% patients were in NYHA class I, 19.5% in II and 1.2% in III. In the mitral position, 222 Sorin prostheses were implanted with the large orifice orientated anteriorly in 46 patients (group 1) and posteriorly-in 176 patients (group 2). Hospital mortality was 4.5% (10/222). Actuarial survival at 66 months was 93.25% +/- 2.1% with a linearized mortality rate of 2.8 patient-years. There were 12 late deaths (9 valve-related and 3 non valve-related). The 66-month actuarial freedom from embolic events was 95.8% +/- 1.89% with a linearized rate of occurrence of 1.44 patient-years.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical scoring systems in meningococcal disease.

Scoring systems used in meningococcal disease have been developed and validated to predict death or severity of illness in cohorts of patients, usually in the setting of intensive care. When using these scores, it is important to remember that any individual's score is only of limited value in prognosticating about that individual patient and can be misleading (1). The accuracy of any score is determined by how well its estimated pattern of mortality compares with that observed in the score's developmental cohort, rather than in individual patients. Scores cannot be considered interchangeable; different scores, comprised of different sets of variables to arrive at the probability of mortality, may arrive at the same overall aggregate risk of mortality in a population of patients with meningococcal disease, despite individual estimates within this cohort of patients differing widely between scores. The ability of a score to predict death for patients who die and survival for those that live is best-described by the area under the receiver operator characteristic (ROC) curve (2). This curve is a plot of sensitivity versus "(1 -specificity)", i.e., the true-positive to false-positive fractions, at different decision thresholds. The greater the discriminative ability of a test, the closer the area under the curve comes towards 1.0, where the true-positive fraction is 1.0 or 100% (perfect specificity) and the false-positive fraction is 0 (perfect sensitivity). No score, however, has sufficient accuracy for individual prognostication and the use of scores in this way is inappropriate for clinical practice or research (3).

On the behaviour of cysteine as ligand of cadmium(II).

The ability of cysteine to form complexes with cadmium(II) in aqueous solutions has been investigated at 25 degrees C and in constant ionic medium NaCl at two different concentrations, 1.00 and 3.00 mol l(-1). The presence of chloride ions was necessary to avoid the precipitation of cadmium(II). Two kinds of measurements were carried out. The electromotive force of galvanic cells containing glass and cadmium amalgam electrodes was measured as a function of cadmium and hydrogen ion concentrations in acid or moderately alkaline solutions in order to obtain the free concentration of cadmium(II) and hydrogen ions. The experimental data obtained in 1.00 mol l(-1) NaCl were explained by assuming the presence of CdHL and CdH(2)L(2), while those obtained in 3.00 mol l(-1) NaCl were accounted for with the formation of CdHL, CdH(2)L(2), CdH(3)L(3) and CdH(2)L(3). Moreover, polarographic measurements were carried out under the same experimental conditions but in alkaline solutions, and the formation of CdL(2) and CdL(3) was assumed from the shift of E(1/2) of cadmium(II) with an excess of cysteine. The stability constants of the assumed species were determined. Protonation constants of cysteine in 1.00 and 3.00 mol l(-1) NaCl have been also determined. A comparison with the behaviour of serine and alpha-aminopropanoate towards cadmium(II) is proposed.

Taurine as a ligand. An investigation of Ag(I)- and Cd(II)-taurine complex equilibria by direct and competitive techniques, and use of the Ag(I)-taurine complex electrode to assess taurine in solution.

The behaviour of taurine as a ligand (L) towards silver(I) and cadmium(II) was studied at 25 degrees C and in 1 mol dm(-3) NaClO(4), as a constant ionic medium. Experimental data, obtained for both cations from electromotive force measurements performed by using silver and cadmium amalgam and glass electrodes, were explained by assuming the formation of the AgL, AgL(2), CdL, and CdL(2) complexes. The taurine protonation constant and stability constants of the above complexes were determined. The cadmium(II)-taurine system was investigated by determining the free concentration of taurine from the Ag electrode potential and the knowledge of equilibria existing between silver(I) and taurine. Experimental data obtained from this approach were explained by assuming the presence of the above species with very close stability constant values. The success of this method supports the possibility of using the Ag/Ag-taurine, taurine electrode to measure the free concentration of taurine in its solutions.

On the acid behaviour of the alcoholic group of serine.

The behaviour of l-serine in strongly alkaline solution was studied at 25 degrees C and in 3.00 mol/l NaClO(4), as a constant ionic medium, to obtain quantitative information about the eventual dissociation of its alcoholic group. A spectrophotometric method was employed to measure the free concentration of hydrogen ions. Two indicators, methyl thymol blue and eriochrome blue type R, were used. Their values as protonation constants and absorbance coefficients were determined in advance. The method here proposed has been validated beforehand by applying it to d-mannite and glycerine and by comparing results so obtained with those previously provided by literature. The investigation showed weak acid properties of the -OH of serine and its dissociation constant was determined. Polynuclear species or further dissociation were not observed.

Long-term results of heart valve replacement with bileaflet prostheses.

Bileaflet cardiac prostheses (St. Jude, CarboMedics, Duromedics, Bicarbon, Jyros) have shown a low incidence of complications and good haemodynamic performance. In the last twelve years, 783 bileaflet prostheses were implanted in 690 patients at our Institution. The population of our study comprises 591 bileaflet prostheses (418 CarboMedics, 124 St. Jude, 49 Bicarbon) implanted in the mitral (MVR) (n = 305) or aortic (AVR), (n = 286) position. The follow-up study evaluated 292 male and 295 female patients with age ranging from 13 and 79 years (mean 50.4 +/- 14.7 years). Hospital mortality was 6.6%. Follow-up was 97% complete, with 1822 +/- 33 patient/years and a mean follow-up of 37 months (range 1 to 144 months). Twelve years actuarial freedom from complication according to prosthetic site were calculated as follows (linearized rates in parentheses): late mortality AVR 97.6% +/- 0.6% (2.3%), MVR 96% +/- 0.5% (2.1%); thrombosis AVR 100%, MVR 96% +/- 0.9% (0.8%); embolism AVR 97% +/- 0.5% (1.5%), MVR 96.6% +/- 0.7% (1.8%). Global freedom from anticoagulant-related haemorrhage was 95% +/- 1.2% (2.3%) and 94.5% +/- 0.7% (2.2%) following AVR, 94 +/- 0.6% (2.1%) following MVR. The difference of the haemorrhagic risk for prosthetic site was not significant (p > 0.05). Functional improvement was confirmed by the low postoperative NYHA functional class. According to our results, cumulative experience with bileaflet valves has shown very good long-term results in term of low rate of complication, long-term survival and quality of life.

Echocardiographic assessment of the subaortic root after implantation of high or low profile mitral prostheses.

In order to evaluate the influence of the profile of mitral prostheses on the outflow tract of the left ventricle, 99 patients underwent M-Mode echocardiography before and after surgery. In 67 cases high profile mitral prostheses had been implanted (48 mechanical and 19 biological); in 32 cases low profile mitral prostheses had been implanted (22 mechanical, 10 biological). Echocardiography showed that the implantation of high profile mitral prostheses did not induce changes of left ventricular diastolic and systolic dimensions ( EDDLV and ESDLV ) while induced significant reduction of left ventricular outflow tract (LVOT) dimension and LVOT/ EDDLV index with subsequent changes in transaortic flow. On the other hand, low profile prostheses did not induce significant changes of left ventricular dimensions, LVOT and LVOT/ EDDLV index. Finally, low profile biological mitral valve prostheses ( Liotta type) induced a significant increase of post-operative dimension of LVOT and LVOT/ EDDLV index. Authors conclude: that the implantation of high profile mitral prostheses changes LVOT dimension and transaortic flow and, that M-Mode echocardiography is a reliable technique to point out these changes.

Stentless fresh pulmonary homograft in mitral position.

We report the implantation of a fresh, stentless pulmonary homograft in the mitral position as an attempt to treat the 2nd recurrence of prosthetic endocarditis in a 66-year-old man. The postoperative course was uneventful; early postoperative transesophageal echocardiography showed a competent valve with a gradient of 4.2 mmHg. Fifteen months postoperatively, transthoracic echocardiography showed a good functioning homograft with a competent valve and no recurrence of endocarditis.