TB-related catastrophic costs in Ethiopia.

OBJECTIVES: To measure the progress towards reducing TB-related catastrophic costs in 19 zones of Amhara, Oromia, SNNP (Southern Nations and Nationalities, and Peoples) and Sidama Regions of Ethiopia. METHODS: A baseline survey was conducted in randomly selected health facilities from all districts within the 19 zones from November 2020 to February 2021. Interventions targeting the major drivers of catastrophic costs identified in the baseline survey, such as installation of 126 GeneXpert and 13 Truenat machines, securing connectivity of 372 GeneXpert, establishing alternative specimen referral systems, and capacity-building of health workers, were implemented. A follow-up survey was conducted from October to December 2022. The WHO generic tool was used to collect data based on probability proportional to size. Data were entered into STATA software, and the proportion of catastrophic costs was calculated and compared between the two surveys. RESULTS: A total of 433 and 397 patients participated in the baseline and follow-up surveys, respectively. The proportion of catastrophic costs reduced from 64.7% to 43.8% (P < 0.0001). The share of direct non-medical costs decreased from 76.2% to 19.2%, while medical and indirect costs increased from 11.6% and 12.3% to 30.4% and 52.4 %. CONCLUSION: The proportion of households facing TB-related catastrophic costs has significantly reduced over the 2-year period. However, it remains unacceptably high and varies among regions. Further reducing the catastrophic costs requires multisectoral response, reviewing the TB service exemption policy, further decentralisation and improving the quality of TB services.

OBJECTIFS: Mesurer les progrès accomplis dans la réduction des coûts catastrophiques liés à la TB dans 19 zones des régions d'Amhara, d'Oromia, de SNNP (Région des nations, nationalités et peuples du Sud) et de Sidama en Éthiopie. MÉTHODES: Une enquête de base a été menée dans des établissements de santé sélectionnés au hasard dans tous les districts des 19 zones de novembre 2020 à février 2021. Des interventions ciblant les principaux facteurs de coûts catastrophiques identifiés dans l'enquête de référence, telles que l'installation de 126 machines GeneXpert et 13 Truenat, la sécurisation de la connectivité de 372 GeneXpert, la mise en place de systèmes alternatifs d'orientation des échantillons et le renforcement des capacités des agents de santé, ont été mises en œuvre. Une enquête de suivi a été menée d'octobre à décembre 2022. L'outil générique de l'OMS a été utilisé pour recueillir des données fondées sur une probabilité proportionnelle à la taille. Les données ont été saisies dans le logiciel STATA, et la proportion des coûts catastrophiques a été calculée et comparée entre les deux enquêtes. RÉSULTATS: Au total, 433 et 397 patients ont participé respectivement à l'enquête de base et à l'enquête de suivi. La proportion des coûts catastrophiques est passée de 64,7% à 43,8% (P < 0,0001). La part des coûts non médicaux directs a diminué, passant de 76,2% à 19,2%, tandis que les coûts médicaux et indirects sont passés de 11,6% et 12,3% à 30,4% et 52,4%. CONCLUSION: La proportion de ménages confrontés à des coûts catastrophiques liés à la tuberculose a considérablement diminué au cours de la période de 2 ans. Cependant, il reste inacceptable et varie selon les régions. Pour réduire davantage les coûts catastrophiques, il faut une réponse multisectorielle, une révision de la politique d'exemption des services de lutte contre la TB, une décentralisation plus poussée et une amélioration de la qualité des services de lutte contre la TB.

Seasonal patterns of tuberculosis case notification in the tropics of Africa: A six-year trend analysis in Ethiopia.

OBJECTIVE: Seasonal variations affect the health system's functioning, including tuberculosis (TB) services, but there is little evidence about seasonal variations in TB case notification in tropical countries, including Ethiopia. This study sought to fill this gap in knowledge using TB data reported from 10 zones, 5 each from Amhara and Oromia regions. METHODS: Notified TB cases for 2010-2016 were analyzed using SPSS version 20. We calculated the quarterly and annual average TB case notification rates and the proportion of seasonal amplitudes. We applied Winters' multiplicative method of exponential smoothing to break down the original time series into seasonal, trend, and irregular components and to build a suitable model for forecasting. RESULTS: A total of 205,575 TB cases were identified (47.8% from Amhara, 52.2% from Oromia), with a male-to-female ratio of 1.2:1. The means of 8,200 (24%), 7,992 (23%), 8,849 (26%), and 9,222 (27%) TB cases were reported during July-September, October-December, January-March, and April-June, respectively. The seasonal component of our model indicated a peak in April-June and a trough in October-December. The seasonal amplitude in Amhara region is 10% greater than that of Oromia (p < 0.05). CONCLUSIONS: TB is shown to be a seasonal disease in Ethiopia, with a peak in quarter four and a low in quarter two of the fiscal year. The peak TB case notification rate corresponds with the end of the dry season in the two agrarian regions of Ethiopia. TB prevention and control interventions, such as efforts to increase community TB awareness about TB transmission and contact tracing, should consider seasonal variation. Regional variations in TB seasonality may require consideration of geographic-specific TB case-finding strategies. The mechanisms underlying the seasonal variation of TB are complex, and further study is needed.

Cognition and neuropathology in aging: multidimensional perspectives from the Rush Religious Orders Study and Rush Memory And Aging Project.

It is increasingly recognized that the correlation between neuropathological lesions and cognition is modest and accounts for about a quarter of the variance of cognition among older adults. Some individuals maintain normal cognitive functioning amidst significant brain pathology, while others suffer varying degrees of cognitive and neurological deterioration that render them dependent and frail. We present data from the Religious Orders Study and the Memory and Aging Project pertaining to pathology and cognition, and propose a paradigm shift in consideration of the neurobiology of healthy aging and dementia. Factors that modify or mediate the association between neuropathology and cognition are also discussed. It is hypothesized that the concept of resilient aging can serve as a useful entity in understanding mechanisms that underlie healthy aging amidst disease-related pathology.

Role of cGMP-dependent protein kinase in regulation of pulmonary vascular smooth muscle cell adhesion and migration: effect of hypoxia.

Exposure to prolonged hypoxia can result in pulmonary vascular remodeling and pulmonary hypertension. Hypoxia induces pulmonary vascular smooth muscle cell (PVSMC) proliferation and vascular remodeling by affecting cell adhesion and migration and secretion of extracellular matrix proteins. We previously showed that acute hypoxia decreases cGMP-dependent protein kinase (PKG) activity in PVSMC and that PKG plays a role in maintaining the differentiated contractile phenotype in normoxia. In this study, we investigated the effect of hypoxia on PVSMC adhesion and migration and the role of PKG in these functions. Ovine fetal pulmonary artery SMC were incubated in normoxia (Po(2) approximately 100 Torr) or hypoxia (Po(2) approximately 30-40 Torr) or treated with the PKG inhibitor DT-3 for 24 h in normoxia. To further study the role of PKG in the modulation of adhesion and migration, PVSMC were transiently transfected with a full-length PKG1alpha [PKG-green fluorescent protein (GFP)] or a dominant-negative construct (G1alphaR-GFP). Cell adhesion to extracellular matrix proteins was determined, and integrin-mediated adhesion was assessed by alpha/beta-integrin-mediated cell adhesion array. Exposure to hypoxia (24 h) and pharmacological inhibition of PKG1 by DT-3 significantly promoted adhesion mediated by alpha(4)-, beta(1)-, and alpha(5)beta(1)-integrins to fibronectin, laminin, and tenacin and also resulted in increased cell migration. Likewise, inhibition of PKG by expression of a dominant-negative PKG1alpha construct increased cell adhesion and migration, comparable to that induced by hypoxia. Dynamic actin reorganization associated with integrin-mediated cell adhesion is partly regulated by the actin-binding protein cofilin, the (Ser3) phosphorylation of which inhibits its actin-severing activity. We found that increased PKG expression and activity is associated with decreased cofilin (Ser3) phosphorylation, implying a role for PKG in the modulation of cofilin activity and actin dynamics. Together, these findings identify cGMP/PKG1 signaling as central to the functional differences between PVSMC exposed to normoxia versus hypoxia.

Risk of dementia in MCI: combined effect of cerebrovascular disease, volumetric MRI, and 1H MRS.

OBJECTIVE: To investigate the combined ability of hippocampal volumes, 1H magnetic resonance spectroscopy (MRS) metabolites, and cerebrovascular disease to predict the risk of progression to dementia in mild cognitive impairment (MCI). METHODS: We identified 151 consecutively recruited subjects with MCI from the Mayo Clinic Alzheimer's Disease Research Center and Patient Registry who underwent MRI and 1H MRS studies at baseline and were followed up with approximately annual clinical examinations. A multivariable proportional hazards model that considered all imaging predictors simultaneously was used to determine whether hippocampal volumes, posterior cingulate gyrus 1H MRS metabolites, white matter hyperintensity load, and presence of cortical and subcortical infarctions are complementary in predicting the risk of progression from MCI to dementia. RESULTS: Seventy-five subjects with MCI progressed to dementia by last follow-up. The model that best predicted progression to dementia included age, sex, hippocampal volumes, N-acetylaspartate (NAA)/creatine (Cr) on 1H MRS, and cortical infarctions. Based on age- and sex-adjusted Kaplan-Meier plots, we estimated that by 3 years, 26% of the MCI patients with normal hippocampal volumes, NAA/Cr ratios >1 SD, and no cortical infarctions will progress to dementia, compared with 78% of the MCI patients with hippocampal atrophy, low NAA/Cr (< or =1 SD), and cortical infarction. CONCLUSIONS: Multiple magnetic resonance (MR) markers of underlying dementia pathologies improve the ability to identify patients with prodromal dementia over a single MR marker, supporting the concept that individuals with multiple brain pathologies have increased odds of dementia compared with individuals with a single pathology.

A plateau in pre-Alzheimer memory decline: evidence for compensatory mechanisms?

OBJECTIVE: To compare logistic and bilogistic models to describe the pattern of cognitive decline in the preclinical phase of Alzheimer disease (AD). METHODS: We conducted mixed effects modeling of Mayo Cognitive Factors Scores to determine the longitudinal pattern of cognitive decline in the period 10 years prior to and 5 years following a clinical diagnosis of AD. Our analysis included 199 people that eventually received a diagnosis of clinically probable AD. Participants had at least two neuropsychological evaluations including one before the evaluation at which they received the AD diagnosis. RESULTS: A bilogistic model, including terms for a plateau in the course of cognitive decline, better fit longitudinal memory scores than a simple logistic model. On average the plateau began about 4 years prior to the clinical diagnosis of AD and ended with a decline that probably contributed to the clinical diagnosis of AD. A similar plateau was not evident in four other cognitive domains. CONCLUSIONS: The current findings may support proposed compensatory hypotheses involving redundant memory systems, up-regulation of neurotransmitters, or recruitment of other neural networks.

Cdk5 mediates changes in morphology and promotes apoptosis of astrocytoma cells in response to heat shock.

The cyclin-dependent kinase member, Cdk5, is expressed in a variety of cell types, but neuron-specific expression of its activator, p35, is thought to limit its activity to neurons. Here we demonstrate that both Cdk5 and p35 are expressed in the human astrocytoma cell line, U373. Cdk5 and p35 are present in the detergent-insoluble cytoskeletal fraction of this cell line and Cdk5 localizes to filopodia and vinculin-rich regions of cell-matrix contact in lamellopodia. When exposed to a 46(o)C heat shock, U373 cells change shape, lose cell-matrix contacts and show increased levels of apoptosis. To test whether Cdk5 activation might play a role in these events, U373 cells were stably transfected with histidine-tagged or green fluorescent protein-tagged constructs of Cdk5 or a dominant negative mutation, Cdk5T33. Under normal growth conditions, growth characteristics of the stably transfected lines were indistinguishable from untransfected U373 cells and Cdk5 localization was not changed. However, when subjected to heat shock, cells stably transfected with Cdk5-T33 remained flattened, showed little loss of cell-matrix adhesion, and exhibited significantly lower levels of apoptosis. In contrast, cells that overexpressed wild-type Cdk5 showed morphological changes similar to those seen in untransfected U373 cells in response to heat shock and had significantly higher levels of apoptosis. Heat-shocked cells showed changes in p35 mobility and stability of the Cdk5/p35 complex consistent with endogenous Cdk5 activity. Together these findings suggest that endogenous Cdk5 activity may play a key role in regulating morphology, attachment, and apoptosis in U373 cells, and raise the possibility that Cdk5 may be a general regulator of cytoskeletal organization and cell adhesion in both neuronal and non-neuronal cells.

Phospholamban remains associated with the Ca2+- and Mg2+-dependent ATPase following phosphorylation by cAMP-dependent protein kinase.

We have used fluorescence and spin-label EPR spectroscopy to investigate how the phosphorylation of phospholamban (PLB) by cAMP-dependent protein kinase (PKA) modifies structural interactions between PLB and the Ca(2+)- and Mg(2+)-dependent ATPase (Ca-ATPase) that result in enzyme activation. Following covalent modification of N-terminal residues of PLB with dansyl chloride or the spin label 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-N-oxyl ('ITC-TEMPO'), we have co-reconstituted PLB with affinity-purified Ca-ATPase isolated from skeletal sarcoplasmic reticulum (SR) with full retention of catalytic function. The Ca(2+)-dependence of the ATPase activity of this reconstituted preparation is virtually identical with that observed using native cardiac SR before and after PLB phosphorylation, indicating that co-reconstituted sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase 1 (SERCA1) and PLB provide an equivalent experimental model for SERCA2a-PLB interactions. Phosphorylation of PLB in the absence of the Ca-ATPase results in a greater amplitude of rotational mobility, suggesting that the structural linkage between the transmembrane region and the N-terminus is destabilized. However, whereas co-reconstitution with the Ca-ATPase restricts the amplitude of rotational motion of PLB, subsequent phosphorylation of PLB does not significantly alter its rotational dynamics. Thus structural interactions between PLB and the Ca-ATPase that restrict the rotational mobility of the N-terminus of PLB are retained following the phosphorylation of PLB by PKA. On the other hand, the fluorescence intensity decay of bound dansyl is sensitive to the phosphorylation state of PLB, indicating that there are changes in the tertiary structure of PLB coincident with enzyme activation. These results suggest that PLB phosphorylation alters its structural interactions with the Ca-ATPase by inducing structural rearrangements between PLB and the Ca-ATPase within a defined complex that modulates Ca(2+)-transport function.

Rearrangement of domain elements of the Ca-ATPase in cardiac sarcoplasmic reticulum membranes upon phospholamban phosphorylation.

Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, and functions to modulate rate-limiting conformational transitions involving the transport activity of the Ca-ATPase. To investigate structural changes within the Ca-ATPase that result from the phosphorylation of PLB by cAMP-dependent protein kinase (PKA), we have covalently bound the long-lived phosphorescent probe erythrosin isothiocyanate (Er-ITC) to cytoplasmic sequences within the Ca-ATPase. Under these labeling conditions, the Ca-ATPase remains catalytically active, indicating that observed changes in rotational dynamics reflect normal conformational transitions. Two major Er-ITC labeling sites were identified using electrospray ionization mass spectrometry (ESI-MS), corresponding to Lys464 and Lys650, which are respectively located within the phosphorylation and nucleotide binding domains of the Ca-ATPase. Frequency-domain phosphorescence measurements of the rotational dynamics of Er-ITC bound to these cytoplasmic sequences within the Ca-ATPase permit the resolution of the dynamic structure of individual domain elements relative to the overall rotational motion of the entire Ca-ATPase polypeptide chain. We observe a significant decrease in the rotational dynamics of Er-ITC bound to the Ca-ATPase upon phosphorylation of PLB by PKA, as evidenced by an increase in the residual anisotropy. These results suggest that phosphorylation of PLB results in a structural reorientation of the phosphorylation or nucleotide binding domains with respect to the membrane normal. In contrast, calcium activation of the Ca-ATPase in the presence of dephosphorylated PLB results in no detectable change in the rotational dynamics of Er-ITC, suggesting that calcium binding and PLB phosphorylation have distinct effects on the conformation of the Ca-ATPase. We suggest that PLB functions to alter the efficiency of phosphoenyzme formation following calcium activation of the Ca-ATPase by modulating the spatial arrangement between ATP bound in the nucleotide binding domain and Asp351 in the phosphorylation domain.

Phosphatidylethanolamine modulates Ca-ATPase function and dynamics.

Phospholipids containing phosphoethanolamine (PE) headgroups within biological membranes have been suggested to be important with respect to the functional regulation of membrane proteins, including the Ca-ATPase in sarcoplasmic reticulum (SR). To investigate the role of PE headgroups in modulating the catalytic activity of the Ca-ATPase, we have reconstituted the Ca-ATPase into unilamellar liposomes containing defined amounts of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC). The enzymatic activity of the Ca-ATPase progressively increases upon incorporation of increasing amounts of PE into reconstituted vesicles, and approaches that characteristic of native SR membranes. To identify structural changes that correlate with enzyme activation, we have used frequency-domain phosphorescence spectroscopy to measure the rotational dynamics of erythrosin isothiocyanate covalently bound to Lys464 in the phosphorylation domain of the Ca-ATPase. Progressive increases in the rotational dynamics of the phosphorylation domain result from the incorporation of increasing amounts of DOPE, and correlate with enhanced enzymatic function. These results suggest that PE headgroups induce dynamic structural rearrangements involving the phosphorylation domain that modify the rates of nucleotide utilization. In contrast, no changes in the rotational dynamics of the lipid acyl chains are observed irrespective of the PE content. Therefore, the enhanced ATP hydrolytic activity associated with the incorporation of DOPE into these proteoliposomes is the result of specific noncovalent interactions involving PE phospholipid headgroups and the Ca-ATPase.

Erythrosin isothiocyanate selectively labels lysine464 within an ATP-protectable binding site on the Ca-ATPase in skeletal sarcoplasmic reticulum membranes.

Conditions that permit the selective modification of an ATP-protectable site on the Ca-ATPase in skeletal sarcoplasmic reticulum (SR) membranes using erythrosin isothiocyanate (Er-ITC) have been identified. The major labeling site for Er-ITC has been identified using reversed-phase HPLC and positive FAB mass spectrometry after exhaustive tryptic digestion of the Er-ITC-modified Ca-ATPase. An ATP-protectable peptide corresponding to M452NVFNTEVRNLSK464VER467 is modified by Er-ITC, the average mass of which is 2830.1 +/- 0.3 Da. The exclusive modification of lysine residues indicates Lys464 as the site of Er-ITC modification. Derivatization with Er-ITC diminishes the secondary activation of steady-state ATPase activity and the rate of dephosphorylation by millimolar concentrations of ATP. In contrast, in the presence of micromolar ATP concentrations Er-ITC modification of the Ca-ATPase does not affect (i) the apparent affinity of ATP, (ii) the maximal extent of phosphoenzyme formation by ATP, (iii) the rate of steady-state ATP hydrolysis, or (iv) the rate of dephosphorylation of the Ca-ATPase. Furthermore, ATP utilization by the Ca-ATPase is unaffected by detergent solubilization, irrespective of Er-ITC modification, indicating that the secondary activation of ATP hydrolysis involves a single Ca-ATPase polypeptide chain. Therefore, Er-ITC does not interfere with the normal structural transitions associated with phosphoenzyme decay. Rather, these results indicate that Er-ITC bound to Lys464 interferes with either ATP binding to a low-affinity site or the associated structural transitions that modulate the rate of enzyme dephosphorylation.

Phosphorylation of phospholamban by cAMP-dependent protein kinase enhances interactions between Ca-ATPase polypeptide chains in cardiac sarcoplasmic reticulum membranes.

We have used spin-label EPR spectroscopy to examine possible alterations in protein-protein interactions that accompany the activation of the cardiac sarcoplasmic reticulum (SR) Ca-ATPase following the phosphorylation of phospholamban (PLB). Using a radioactive derivative of a maleimide spin label (MSL), we have developed conditions for the selective spin-labeling of the Ca-ATPase in both native cardiac and skeletal sarcoplasmic reticulum membranes. The rotational dynamics of the cardiac and skeletal Ca-ATPase isoforms in native SR membranes were measured using saturation transfer EPR. We report that the phosphorylation of PLB in cardiac SR results in a (1.8 +/- 0.2)-fold reduction in the overall rotational mobility of the Ca-ATPase. The alteration in the rotational dynamics of the Ca-ATPase is the direct result of the phosphorylation of PLB, and is not related to the phosphorylation of the Ca-ATPase or any other SR proteins since no alteration in the ST-EPR spectrum is observed as a result of conditions that phosphorylate the cardiac Ca-ATPase with ATP. Neither do the use of conditions that activate the Ca-ATPase in cardiac SR result in the alteration of the rotational dynamics or catalytic properties of the Ca-ATPase in skeletal SR where PLB is not expressed. Measurements of the rotational dynamics of stearic acid spin labels (SASL) incorporated into cardiac SR membranes with a nitroxide at the 5- and 12-positions using conventional EPR indicate that there is virtually no difference in the lipid acyl chain dynamics in cardiac SR membranes upon the phosphorylation of PLB. These results indicate that the decrease in the rotational dynamics of the Ca-ATPase in cardiac SR membranes associated with the phosphorylation of PLB is related to enhanced interactions between individual Ca-ATPase polypeptide chains due to (i) an alteration in the spatial arrangement of cardiac Ca-ATPase polypeptide chains within a defined oligomeric state or (ii) increased protein-protein associations. We suggest that altered interactions between Ca-ATPase polypeptide chains and PLB serves to modulate the activation barrier associated with calcium activation of the Ca-ATPase in cardiac SR membranes.