Inhibition of 26S proteasome activity by α-synuclein is mediated by the proteasomal chaperone Rpn14/PAAF1.

Parkinson's disease (PD) is characterized by aggregation of α-synuclein (α-syn) into protein inclusions in degenerating brains. Increasing amounts of aggregated α-syn species indicate significant perturbation of cellular proteostasis. Altered proteostasis depends on α-syn protein levels and the impact of α-syn on other components of the proteostasis network. Budding yeast Saccharomyces cerevisiae was used as eukaryotic reference organism to study the consequences of α-syn expression on protein dynamics. To address this, we investigated the impact of overexpression of α-syn and S129A variant on the abundance and stability of most yeast proteins using a genome-wide yeast library and a tandem fluorescent protein timer (tFT) reporter as a measure for protein stability. This revealed that the stability of in total 377 cellular proteins was altered by α-syn expression, and that the impact on protein stability was significantly enhanced by phosphorylation at Ser129 (pS129). The proteasome assembly chaperone Rpn14 was identified as one of the top candidates for increased protein stability by expression of pS129 α-syn. Elevated levels of Rpn14 enhanced the growth inhibition by α-syn and the accumulation of ubiquitin conjugates in the cell. We found that Rpn14 interacts physically with α-syn and stabilizes pS129 α-syn. The expression of α-syn along with elevated levels of Rpn14 or its human counterpart PAAF1 reduced the proteasome activity in yeast and in human cells, supporting that pS129 α-syn negatively affects the 26S proteasome through Rpn14. This comprehensive study into the alternations of protein homeostasis highlights the critical role of the Rpn14/PAAF1 in α-syn-mediated proteasome dysfunction.

A novel digitonin/graphene oxide/iron oxide nanocomposite: synthesis, physiochemical characterization and antioxidant activity.

In this work, iron oxide (Fe3O4) magnetic nanoparticles (MNPs) and graphene oxide (GO) nanosheets were prepared via the co-precipitation technique and the Modified Hummer method. Fe3O4 MNPs and GO nanosheets were combined to prepare Fe3O4/GO nanocomposite and subsequently conjugated with Digitonin (DIG) in order to obtain a dual-targeted delivery system based on DIG/Fe3O4/GO nanocomposite. SEM images reveal the presence of Fe3O4 MNPs at a scale of 100 nm, exhibiting dispersion between the GO nanosheets. Aggregation of the DIG/Fe3O4/GO nanocomposite was observed at various size scales. The XRD structural analysis confirms the crystal structure of the prepared samples. The Fe3O4 MNPs demonstrated the main XRD-diffracted peaks. Also, GO nanosheets exhibit crystalline characteristics on the (001) and (002) planes. The predominant peaks observed in the DIG/GO/Fe3O4 nanocomposite are attributed to the crystal phases of Fe3O4 MNPs. The FT-IR vibrational modes observed in the GO/DIG/Fe3O4 nanocomposite indicate the presence of crosslinking between GO nanosheet layers and the Fe3O4 MNPs. The antioxidant activity of the prepared samples was measured and the DIG/GO/Fe3O4 nanocomposite demonstrated a significantly high antioxidant activity in both 2-diphenyl-1-picrylhydrazyl (DPPH·) and 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS·+) tests.

Nanofractionation spotter technology for rapid contactless and high-resolution deposition of LC eluent for further off-line analysis.

The development of a contactless postcolumn spotter technology capable of rapidly and accurately depositing LC eluent onto another platform (e.g., 1536-well microtiter plates) is described. Many detection methodologies are suitable for online analysis, such as mass spectrometry, UV-vis, and fluorescence. In some cases, when online analysis is less suitable, off-line postcolumn analysis is the methodology of choice and usually relies on LC-based fractionation prior to detection (e.g., MALDI-MS, Raman spectrsocopy, biochemical assays). As fractionation generally involves loss in resolution, the technology described here allows high-resolution contactless fractionation by tailoring the fractionation frequency to the chromatographic peaks and mixing in of postcolumn reagents. Droplet ejection at frequencies of at least 6 Hz could be performed in the nanoliter to low microliter range with repeatabilities of ∼6%. Furthermore, multiple droplets can be ejected at the same position thereby allowing adjustment of fractionation volume and speed. The technology was evaluated, optimized, and validated prior to two proof-of-principle demonstrations comprising off-line chemical detection of injected fluorescein and off-line postcolumn biochemical detection of acetylcholine-binding protein ligands, both based on 1536-well plate reader analysis.

Anaphylactic and nonanaphylactic reactions to SARS-CoV-2 vaccines: a systematic review and meta-analysis.

BACKGROUND: Currently there is no systematic review and meta-analysis of the global incidence rates of anaphylactic and nonanaphylactic reactions to SARS-CoV-2 vaccines in the general adult population. OBJECTIVES: To estimate the incidence rates of anaphylactic and nonanaphylactic reactions after COVID-19 vaccines and describe the demographic and clinical characteristics, triggers, presenting signs and symptoms, treatment and clinical course of confirmed cases. DESIGN: A systematic review and meta-analysis. Preferred Reporting Items for Systematic Reviews and Meta-Analyses [PRISMA] statement was followed. METHODS: Electronic databases (Proquest, Medline, Embase, Pubmed, CINAHL, Wiley online library, and Nature) were searched from 1 December 2020 to 31 May 2021 in the English language using the following keywords alone or in combination: anaphylaxis, non-anaphylaxis, anaphylactic reaction, nonanaphylactic reaction, anaphylactic/anaphylactoid shock, hypersensitivity, allergy reaction, allergic reaction, immunology reaction, immunologic reaction, angioedema, loss of consciousness, generalized erythema, urticaria, urticarial rash, cyanosis, grunting, stridor, tachypnoea, wheezing, tachycardia, abdominal pain, diarrhea, nausea, vomiting and tryptase. We included studies in adults of all ages in all healthcare settings. Effect sizes of prevalence were pooled with 95% confidence intervals (CIs). To minimize heterogeneity, we performed sub-group analyses. RESULTS: Of the 1,734 papers that were identified, 26 articles were included in the systematic review (8 case report, 5 cohort, 4 case series, 2 randomized controlled trial and 1 randomized cross-sectional studies) and 14 articles (1 cohort, 2 case series, 1 randomized controlled trial and 1 randomized cross-sectional studies) were included in meta-analysis. Studies involving 26,337,421 vaccine recipients [Pfizer-BioNTech (n = 14,505,399) and Moderna (n = 11,831,488)] were analyzed. The overall pooled prevalence estimate of anaphylaxis to both vaccines was 5.0 (95% CI 2.9 to 7.2, I2 = 81%, p = < 0.0001), while the overall pooled prevalence estimate of nonanaphylactic reactions to both vaccines was 53.9 (95% CI 0.0 to 116.1, I2 = 99%, p = < 0.0001). Vaccination with Pfizer-BioNTech resulted in higher anaphylactic reactions compared to Moderna (8.0, 95% CI 0.0 to 11.3, I2 = 85% versus 2.8, 95% CI 0.0 to 5.7, I2 = 59%). However, lower incidence of nonanaphylactic reactions was associated with Pfizer-BioNTech compared to Moderna (43.9, 95% CI 0.0 to 131.9, I2 = 99% versus 63.8, 95% CI 0.0 to 151.8, I2 = 98%). The funnel plots for possible publication bias for the pooled effect sizes to determine the incidence of anaphylaxis and nonanaphylactic reactions associated with mRNA COVID-19 immunization based on mRNA vaccine type appeared asymmetrical on visual inspection, and Egger's tests confirmed asymmetry by producing p values < 0.05. Across the included studies, the most commonly identified risk factors for anaphylactic and nonanaphylactic reactions to SARS-CoV-2 vaccines were female sex and personal history of atopy. The key triggers to anaphylactic and nonanaphylactic reactions identified in these studies included foods, medications, stinging insects or jellyfish, contrast media, cosmetics and detergents, household products, and latex. Previous history of anaphylaxis; and comorbidities such as asthma, allergic rhinitis, atopic and contact eczema/dermatitis and psoriasis and cholinergic urticaria were also found to be important. CONCLUSION: The prevalence of COVID-19 mRNA vaccine-associated anaphylaxis is very low; and nonanaphylactic reactions occur at higher rate, however, cutaneous reactions are largely self-limited. Both anaphylactic and nonanaphylactic reactions should not discourage vaccination.

Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays.

One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC(50) values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture.

Isolation of high density lipoprotein subclasses by electrofiltration and their chemical components.

The exact role of high density lipoprotein in atheroprotection is not well understood yet due to its complex nature; it comprises more than ten subclasses that vary in size, composition, and function. Isolation and characterization of these subclasses is an important step for further studies addressing their functions in health and disease. In this work, we present a novel method that is relatively simple and efficient for isolation of high density lipoprotein subclasses. The method depends on fractional filtration of the subclasses through a preformed gel membrane system under the effect of an electric field, where the stepwise isolation of the subclasses depends on differences in their rates of migration in polyacrylamide gel. Using this design, we were able to isolate seven high density lipoprotein subclasses with relative molecular masses of 42,000-50,000; 71,000; 103,000; 124,000; 150,000; 182,000; and 219,000. All the subclasses contained apolipoprotein A-I, phosphatidylcholine, sphingomyelin, free cholesterol, esterified cholesterol, and triacylglycerols. Some fractions of some samples contained the apolipoproteins A-II, C-I, C-II, C-III, and E. A subclass of molecular mass of 106,000 was identified and isolated from a healthy young subject that contained albumin and apoA-I with some free and esterified cholesterol, but with no triacylglycerols. This electrofiltration technique offers a novel tool for isolating pure native high density lipoprotein subclasses in a concentrated form that can be used directly for detailed studies of their physicochemical and physiological properties.

Na+/H+ antiport activity and cell growth in cultured skin fibroblasts of IDDM patients with nephropathy.

IDDM patients with incipient and overt nephropathy have been found to exhibit an overactivity of RBC sodium-lithium countertransport. To explore the physiological relevance of this finding, we measured the activity of Na+/H+ antiport in serially passaged cultured skin fibroblasts from IDDM patients with and without nephropathy and from normal, nondiabetic control subjects. Na+/H+ antiport activity (measured as the rate of amiloride-sensitive Na+ influx at pHi = 6.4, extracellular pH = 8.0, and [Na+] = 1 mM) was elevated significantly in IDDM patients with nephropathy compared with IDDM patients without nephropathy and nondiabetic control subjects (13.35 +/- 3.8 vs. 8.54 +/- 2.0 vs. 7.33 +/- 2.3 nmol Na+.mg protein-1.min-1; P less than 0.006 and P less than 0.001, respectively). A kinetic analysis of Na+/H+ antiport activity showed that the raised activity in IDDM patients with nephropathy was caused by an increased Vmax for extracellular Na+. Km values were similar in the three groups. pH-stimulated amiloride-sensitive Na+ influx also was higher under baseline conditions and after serum stimulation in cells from IDDM patients with nephropathy. pHi values were significantly higher, both during active proliferation and after 10-min exposure to serum, in cells from IDDM patients with nephropathy, compared with IDDM patients without nephropathy and nondiabetic control subjects. Serum-stimulated incorporation of [3H]thymidine into DNA was greater in IDDM patients with nephropathy than in the other two groups (35.7 +/- 18.9- vs. 17.4 +/- 7.5- vs. 11.9 +/- 8.7-fold stimulation above baseline; P less than 0.01 for both.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-lithium countertransport in microalbuminuric insulin-dependent diabetic patients.

A familial predisposition to arterial hypertension has recently been suggested as one important component of the susceptibility to diabetic kidney disease. Sodium-lithium countertransport activity, a marker of risk for essential hypertension, has been found to be increased in diabetic patients with overt nephropathy. We have measured red blood cell sodium-lithium counter-transport activity in 36 microalbuminuric insulin-dependent diabetic patients, a group at high risk of progression to clinical nephropathy and cardiovascular disease, and compared it with that of a matched group of 36 normoalbuminuric diabetic patients. Sodium-lithium countertransport was higher in the microalbuminuric (0.43 [95% confidence interval (CI) 0.38-0.47] mmol/l red blood cells [RBC]/hr) than in the normoalbuminuric diabetic patients (0.29 [0.25-0.33] mmol/l RBC/hr, mean difference 0.14 [0.08-0.20]; p less than 0.0001). Microalbuminuric patients had a higher frequency of parental hypertension than normoalbuminuric diabetic patients (56% vs. 28%, p less than 0.05). Sodium-lithium countertransport was related to mean arterial pressure in the microalbuminuric patients (r = 0.54, p less than 0.001) and to daily insulin requirements in both groups (microalbuminuric patients r = 0.39, p less than 0.05; normoalbuminuric patients r = 0.42, p less than 0.01). In a subset of patients in whom lipoproteins were measured, sodium-lithium countertransport activity was related to total and very low density lipoprotein triglycerides (r = 0.41, p less than 0.05 and r = 0.48, p less than 0.05) and to apolipoprotein B (r = 0.56, p less than 0.05), independently of body mass index, albumin excretion rate, glycemic control, and insulin dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-lithium countertransport activity in red cells of patients with insulin dependent diabetes and nephropathy and their parents.

OBJECTIVE: To determine whether there are familial and genetic aspects of sodium-lithium countertransport activity in red cells in diabetic nephropathy. DESIGN: Case-control study. SETTING: Teaching hospital diabetic clinic. SUBJECTS: 40 Patients with insulin dependent diabetes, both of whose parents were alive: 20 with persistent proteinuria and 20 with normal albumin excretion matched for age, duration of diabetes, and body mass index. All 80 parents. MAIN OUTCOME MEASURES: Sodium-lithium countertransport activity in red cells and arterial blood pressure. RESULTS: Sodium-lithium countertransport activity in red cells was higher in the patients with proteinuria than in the patients with normoalbuminuria (mean (95% confidence interval) 0.47 (0.39 to 0.54) v 0.33 (0.28 to 0.38) mmol/l red cells/h respectively, p = 0.0036; mean difference 0.14 (0.04 to 0.22)). The mean countertransport activity for the two parents of each patient was calculated, and from this the mean value for each group of parents was calculated; the value was higher in the parents of the patients with proteinuria than in the parents of the patients with normoalbuminuria (0.40 (0.32 to 0.48) v 0.30 (0.26 to 0.33) mmol/l red cells/h respectively, p = 0.016; 0.10 (0.02 to 0.19)). Twenty-eight of the parents of the patients with proteinuria compared with 12 of the parents of the patients with normoalbuminuria had a countertransport activity that was above the median value in all 80 parents (p less than 0.001). Mean arterial blood pressure in the parents of the patients with proteinuria was related to that of their offspring (r = 0.46; p less than 0.01). There was a positive correlation between the sodium-lithium countertransport activity in red cells in the parents and their offspring when all parents and patients were considered (r = 0.37; p less than 0.001). CONCLUSIONS: Increased sodium-lithium countertransport activity in red cells in the parents of diabetic patients with nephropathy provides further evidence that familial, and possibly genetic, factors related to a predisposition to arterial hypertension have a role in the susceptibility of diabetic renal disease.

Evaluation of the role of craniotomy in developing countries.

A review of 32 cases of craniotomy performed at the Jinnah Postgraduate Medical Centre Karachi, over a period of 6 years from January, 1984 to December, 1989 is presented. Total number of deliveries during this period were 37,682. Frequency of craniotomy was 0.08% i.e., 1 in 1177 deliveries. Of these 32 craniotomies, 28 were done on non-deformed dead fetus, 16 because of obstructed labour, 7 in cases of after coming head of breech and 5 in failure to progress in second stage of labour. Four cases were of live hydrocephalic fetus in whom cephalocentesis was done followed by craniotomy due to failure to progress in labour. Maternal morbidity was 12.5% with no maternal death. We conclude that it is safe and quicker than caesarean section in selected cases. Though unpleasant to perform, is of great relief to the patient and her family.

Culdocentesis in diagnosis of disturbed ectopic pregnancy still a useful procedure in developing countries.

Over a period of 5 years culdocentesis was carried out in 156 cases of suspected ectopic pregnancy using needle aspiration through the pouch of Douglas. The result was positive in 134 cases, with 131 being true positive and 3 false positive. In 22 cases the result was negative, 6 of which were false negative. It is concluded that culdocentesis is an effective method of diagnosing disturbed ectopic pregnancy.

Role of colposcopy in the diagnosis and outpatient treatment of cervical intraepithelial neoplasia.

One hundred and seventy nine women with abnormal smears referred for colposcopy to the Samaritan Hospital were studied. After confirming CIN on repeat smear, colposcopy and histology, cone biopsy was carried out. One hundred and twenty one patients (68%) had treatment either with electrical loop diathermy under local anaesthesia (105 patients, 59%) or laser excision of transformation zone under general anaesthesia as a day case (16 patients, 9%). Cone biopsy was done in 121 patients (32%). We conclude that colposcopy is a valuable tool in combination with cytology and histology for diagnosis and delineating the extent of CIN, thus avoiding unnecessary cone biopsies particularly in women of child bearing age. The majority of patients were treated by an outpatient procedure, which is easy to learn, safe, effective and inexpensive.

Separation of biomacromolecules by electrofiltration through gel layers.

A straightforward method for concomitant separation and isolation of biomacromolecules from a mixture in solution was developed. Three gel layers that comprise a middle separation layer of 10% polyacrylamide gel were constructed. This gel system was formed in an electroconcentration apparatus above a collection chamber surrounded at the bottom by a dialysis membrane. The mixture is applied over the gel layers where biomacromolecules are caused to migrate by electrophoresis through the gel system, where they are separated into discrete bands and electroeluted into the collection chamber without dismantling the apparatus. The isolated biomacromolecules are removed from the chamber in a highly pure and concentrated form ready for further investigations. Cooling can be applied throughout the whole process, and the setup and conditions of run can be modified according to the characteristics of the biomacromolecules to be purified. The components of a mixture containing the glycoprotein ovalbumin and bovine serum albumin monomer, dimer, and tetramer were successfully isolated as concentrated and highly pure fractions with good recoveries ranging from 70 to 89%. Other proteins were successfully isolated under denaturing conditions in the presence of sodium dodecyl sulfate (SDS) or 6 M urea.

Prevalence of raised sodium-lithium countertransport activity in type 1 diabetic patients.

The prevalence of raised Na+/Li+ countertransport (CT) activity (greater than 0.41 mmol/liter RBC/hr) was assessed in 185 consecutive insulin-dependent diabetic patients attending an outpatient diabetic clinic. Normoalbuminuria was defined as an overnight albumin excretion rate (AER) of less than 20 micrograms/min (N = 121), microalbuminuria as AER between 20 and 150 micrograms/min (N = 35) and macroalbuminuria as AER greater than or equal to 150 micrograms/min (N = 29). The prevalence of elevated Na+/Li+CT (greater than 0.41 mmol/liter RBC/hr) was 21.5, 42.8 and 51.7% (P = 0.0005), in patients with normo-, micro- and macroalbuminuria, respectively. In the whole group, Na+/Li+CT was significantly related to mean blood pressure (MBP; rs = 0.37, P less than 0.001) and AER (rs = 0.38, P less than 0.001). In a multiple regression analysis the significant correlates of AER, as a continuous variable, or of proteinuria (micro + macroalbuminuria), as a categorical variable, were Na+/Li+CT, MBP, duration of diabetes and glycosylated hemoglobin (HbA1). The frequency of normoalbuminuric patients with high Na+/Li+CT activity fell with duration of diabetes. The risk of proteinuria was significantly greater in patients with raised Na+/Li+CT compared to those with Na+/Li+CT within the normal range (odds ratio 3.8, 95% CI, 1.9 and 7.8). A relative excess of patients with proteinuria (micro + macroalbuminuria) was found in the group with elevated Na+/Li+CT and HbA1 above the median value (8.05%) of the whole population (chi 2 = 9.7, P less than 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-lithium countertransport activity as a determinant of deterioration of glomerular function in IgA nephropathy.

We measured Na+/Li+ CT in 16 IgA nephropathy patients. Records were reviewed (mean observation period 5.5 years) for serial measurements of blood pressure (BP), urinary protein excretion, GFR (51Cr-EDTA) and plasma creatinine. Na+/Li+ CT correlated with the slope of the plot of GFR versus time (rs = -0.66, p = 0.005) systolic BP at diagnosis (rs = 0.62, p = 0.011) and both systolic and diastolic BP at the end of follow-up (rs = 0.69, p = 0.003, and rs = 0.56, p = 0.023). A diastolic blood pressure (DBP) > or = 95 mm Hg was associated with a faster rate of GFR decline (rate of change of GFR: -0.40 vs. -0.14 ml/min/month, p = 0.07; for DBP > or = 95 vs. < 95 mm Hg, respectively). In a multiple regression analysis with the rate of decline of GFR as dependent variable, Na+/Li+ CT emerged as a significant and independent determinant of the rate of fall of GFR (beta coefficient -1.56, SE beta 0.49, p = 0.006) and explained 52.7% of the variation in the GFR fall. Higher activities of Na+/Li+ CT are significantly associated with an increased rate of deterioration of renal function in IgA nephropathy; part of this effect could be mediated by higher blood pressure values.