NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse.

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

Accessible and reliable neurometric testing in humans using a smartphone platform.

Tests of human brain circuit function typically require fixed equipment in lab environments. We have developed a smartphone-based platform for neurometric testing. This platform, which uses AI models like computer vision, is optimized for at-home use and produces reproducible, robust results on a battery of tests, including eyeblink conditioning, prepulse inhibition of acoustic startle response, and startle habituation. This approach provides a scalable, universal resource for quantitative assays of central nervous system function.

TB in the elderly: clinical features and outcomes.

BACKGROUND: TB in low-incidence countries is characterised by changes in age distribution towards larger numbers of cases among the elderly.OBJECTIVES: To investigate clinical features and outcomes of TB treatment in older patients and identify predictors of poor outcome.METHODS: Multicentre retrospective study of new TB cases from 53 hospitals included in the registry of the Integrated Tuberculosis Research Programme of the Spanish Society of Pulmonology and Thoracic Surgery (Sociedad Española de Neumología y Cirugía Torácica) between 2006 and 2020.RESULTS: We identified 731 patients aged ≥75 years from a cohort of 7,505 patients with TB. In the elderly, weight loss, disseminated disease and normal X-rays or infiltrates without cavitation were more common. All-cause mortality was 16% (5% of deaths due to TB). The elderly had higher rates of toxicity (6.7%) and hospital admissions (36%). In the multivariate analysis of predictors of TB mortality in ≥75-year-olds, only weight, age and treatment with non-standard regimens remained significant.CONCLUSIONS: TB in older patients needs more attention and remains a challenge because of a lack of specific clinical and radiological features. Standard treatment is effective, although mortality is higher than in young patients. Low weight, non-standard regimens and age are significant predictors of TB mortality.

The anion exchanger Ae2 is required for enamel maturation in mouse teeth.

One of the mechanisms by which epithelial cells regulate intracellular pH is exchanging bicarbonate for Cl(-). We tested the hypothesis that in ameloblasts the anion exchanger-2 (Ae2) is involved in pH regulation during maturation stage amelogenesis. Quantitative X-ray microprobe mineral content analysis, scanning electron microscopy, histology, micro-computed tomography and Ae2 immuno-localisation analyses were applied to Ae2-deficient and wild-type mouse mandibles. Immuno-localisation of Ae2 in wild-type mouse incisors showed a very strong expression of Ae2 in the basolateral membranes of the maturation stage ameloblasts. Strikingly, zones of contiguous ameloblasts were found within the maturation stage in which Ae2 expression was extremely low as opposed to neighbouring cells. Maturation stage ameloblasts of the Ae2(a,b)(-/-) mice failed to stain for Ae2 and showed progressive disorganisation as enamel development advanced. Maturation stage enamel of the Ae2(a,b)(-/-) mice contained substantially less mineral and more protein than wild-type enamel as determined by quantitative X-ray microanalysis. Incisor enamel was more severely affected than molar enamel. Scanning electron microscopy revealed that the rod-inter-rod structures of the Ae2(a,b)(-/-) mice incisor enamel were absent. Mineral content of dentine and bone of Ae2(a,b)(-/-) mice was not significantly different from wild-type mice. The enamel from knockout mouse teeth wore down much faster than that from wild-type litter mates. Basolateral bicarbonate secretion via the anionic exchanger Ae2 is essential for mineral growth in the maturation stage enamel. The observed zonal expression of Ae2 in the maturation stage ameloblasts is in line with a model for cyclic proton secretion during maturation stage amelogenesis.

Computer simulation of cerebellar information processing.

Although many functions have been ascribed to the cerebellum, the uniformity of its synaptic organization suggests that a single, characteristic computation may be common to all. Computer simulations are useful in examining this cerebellar computation, as they inherently address function at the level of information processing. Progress is facilitated by factors that make the cerebellum particularly amenable to such analysis. We review progress from two contrasting approaches. Top-down simulations begin with hypotheses about computational mechanisms and then ask how such mechanisms might operate within the cerebellum. Bottom-up simulations attempt to build a representation of the cerebellum that reflects known cellular and synaptic components as accurately as possible. We describe recent advances from these two approaches that are leading to an understanding of what information the cerebellum processes and how its neurons and synapses accomplish this task.

Cerebellar function: coordination, learning or timing?

Theories of cerebellar function have largely involved three ideas: movement coordination, motor learning or timing. New evidence indicates these distinctions are not particularly meaningful, as the cerebellum influences movement execution by feedforward use of sensory information via temporally specific learning.

Mechanisms of cerebellar learning suggested by eyelid conditioning.

Classical eyelid conditioning has been used to great advantage in demonstrating that the cerebellum helps to improve movements through experience, and in identifying the underlying mechanisms. Results from recent studies support the hypotheses that learning occurs in both the cerebellar nucleus and cortex, and that these sites make different contributions. Specifically, results indicate that the cerebellar cortex is responsible for temporally specific learning. A combination of experimental and computational studies has been important for arriving at these conclusions, which seem to be applicable to the broad range of movements to which the cerebellum contributes.

Factors Associated with Fatality during the Intensive Phase of Anti-Tuberculosis Treatment.

OBJECTIVE: To determine the case-fatality rate (CFR) at the end of the intensive phase of tuberculosis (TB) treatment, and factors associated with fatality. METHODS: TB patients diagnosed between 2006 and 2013 were followed-up during treatment. We computed the CFR at the end of the intensive phase of TB treatment, and the incidence of death per 100 person-days (pd) of follow-up. We performed survival analysis using the Kaplan-Meier method and Cox regression, and calculate hazard ratios (HR) and 95% confidence intervals (CI). RESULTS: A total of 5,182 patients were included, of whom 180 (3.5%) died; 87 of these deaths (48.3%) occurred during the intensive phase of treatment, with a CFR of 1.7%. The incidence of death was 0.028/100 pd. The following factors were associated with death during the intensive phase: being >50 years (HR = 36.9;CI:4.8-283.4); being retired (HR = 2.4;CI:1.1-5.1); having visited the emergency department (HR = 3.1;CI:1.2-7.7); HIV infection (HR = 3.4;CI:1.6-7.2); initial standard treatment with 3 drugs (HR = 2.0;CI:1.2-3.3) or non-standard treatments (HR = 2.68;CI:1.36-5.25); comprehension difficulties (HR = 2.8;CI:1.3-6.1); and smear-positive sputum (HR = 2.3-CI:1.0-4.8). CONCLUSION: There is a non-negligible CFR during the intensive phase of TB, whose reduction should be prioritised. The CFR could be a useful indicator for evaluating TB programs.

Simulations of cerebellar motor learning: computational analysis of plasticity at the mossy fiber to deep nucleus synapse.

We question the widely accepted assumption that a molecular mechanism for long-term expression of synaptic plasticity is sufficient to explain the persistence of memories. Instead, we show that learning and memory require that these cellular mechanisms be correctly integrated within the architecture of the neural circuit. To illustrate this general conclusion, our studies are based on the well characterized synaptic organization of the cerebellum and its relationship to a simple form of motor learning. Using computer simulations of cerebellar-mediated eyelid conditioning, we examine the ability of three forms of plasticity at mossy fiber synapses in the cerebellar nucleus to contribute to learning and memory storage. Results suggest that when the simulation is exposed to reasonable patterns of "background" cerebellar activity, only one of these three rules allows for the retention of memories. When plasticity at the mossy fiber synapse is controlled by nucleus or climbing fiber activity, the circuit is unable to retain memories because of interactions within the network that produce spontaneous drift of synaptic strength. In contrast, a plasticity rule controlled by the activity of the Purkinje cell allows for a memory trace that is resistant to ongoing activity in the circuit. These results suggest specific constraints for theories of cerebellar motor learning and have general implications regarding the mechanisms that may contribute to the persistence of memories.

A mechanism for savings in the cerebellum.

The phenomenon of savings (the ability to relearn faster than the first time) is a familiar property of many learning systems. The utility of savings makes its underlying mechanisms of special interest. We used a combination of computer simulations and reversible lesions to investigate mechanisms of savings that operate in the cerebellum during eyelid conditioning, a well characterized form of motor learning. The results suggest that a site of plasticity outside the cerebellar cortex (possibly in the cerebellar nucleus) can be protected from the full consequences of extinction and that the residual plasticity that remains can later contribute to the savings seen during relearning.

Leukotriene A4: metabolism in different rat tissues.

The transformation of leukotriene A4 into dihydroxyeicosatetraenoic acids and sulfidopeptide leukotrienes was determined in homogenates of rat tissues supplied with glutathione and albumin. The highest production of leukotriene B4 was found in spleen, lung and small intestine, while leukotriene C4 dominated in liver and lung. 5(S),6(R)-Dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5,6-DHETE) was formed in all tissues, most prominently in kidney, heart and brain. We also found another isomer of 5,6-dihydroxyeicosatetraenoic acid produced in the kidney. This compound was derived from 5,6-DHETE by isomerization, probably of the 11-cis double bond to 11-trans, and the process appeared to be catalyzed by a membrane-bound factor.

Recombinant mouse leukotriene A4 hydrolase: a zinc metalloenzyme with dual enzymatic activities.

Recombinant mouse leukotriene A4 hydrolase was expressed in Escherichia coli as a fusion protein with ten additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was shown to contain one mol of zinc/mol of enzyme. Apparent kinetic constants (Km and Vmax) for the conversion of leukotriene A4 to leukotriene B4 (at 0 degree C, pH 8) were 5 microM and 900 nmol/mg per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine-4-nitroanilide. Km and Vmax for this reaction (at 37 degrees C, pH 8) were 680 microM and 365 nmol/mg per min, respectively. Apo-leukotriene A4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt. Exposure of the enzyme to leukotriene A4 resulted in a dose-dependent inactivation of both enzyme activities.

Ameloblast Modulation and Transport of Cl⁻, Na⁺, and K⁺ during Amelogenesis.

Ameloblasts express transmembrane proteins for transport of mineral ions and regulation of pH in the enamel space. Two major transporters recently identified in ameloblasts are the Na(+)K(+)-dependent calcium transporter NCKX4 and the Na(+)-dependent HPO4 (2-) (Pi) cotransporter NaPi-2b. To regulate pH, ameloblasts express anion exchanger 2 (Ae2a,b), chloride channel Cftr, and amelogenins that can bind protons. Exposure to fluoride or null mutation of Cftr, Ae2a,b, or Amelx each results in formation of hypomineralized enamel. We hypothesized that enamel hypomineralization associated with disturbed pH regulation results from reduced ion transport by NCKX4 and NaPi-2b. This was tested by correlation analyses among the levels of Ca, Pi, Cl, Na, and K in forming enamel of mice with null mutation of Cftr, Ae2a,b, and Amelx, according to quantitative x-ray electron probe microanalysis. Immunohistochemistry, polymerase chain reaction analysis, and Western blotting confirmed the presence of apical NaPi-2b and Nckx4 in maturation-stage ameloblasts. In wild-type mice, K levels in enamel were negatively correlated with Ca and Cl but less negatively or even positively in fluorotic enamel. Na did not correlate with P or Ca in enamel of wild-type mice but showed strong positive correlation in fluorotic and nonfluorotic Ae2a,b- and Cftr-null enamel. In hypomineralizing enamel of all models tested, 1) Cl(-) was strongly reduced; 2) K(+) and Na(+) accumulated (Na(+) not in Amelx-null enamel); and 3) modulation was delayed or blocked. These results suggest that a Na(+)K(+)-dependent calcium transporter (likely NCKX4) and a Na(+)-dependent Pi transporter (potentially NaPi-2b) located in ruffle-ended ameloblasts operate in a coordinated way with the pH-regulating machinery to transport Ca(2+), Pi, and bicarbonate into maturation-stage enamel. Acidification and/or associated physicochemical/electrochemical changes in ion levels in enamel fluid near the apical ameloblast membrane may reduce the transport activity of mineral transporters, which results in hypomineralization.

On-water remote monitoring robotic system for estimating the patch coverage of Anabaena sp. filaments in shallow water.

An on-water remote monitoring robotic system was developed for indirectly estimating the relative density of marine cyanobacteria blooms at the subtidal sandy-rocky beach in Balandra Cove, Baja California Sur, Mexico. The system is based on an unmanned surface vehicle to gather underwater videos of the seafloor for avoiding physical damage on Anabaena sp. cyanobacteria colonies, which grow in tufts of filaments weakly attached to rocks, seagrass, and macroalgae. An on-axis image stabilization mechanism was developed to support a camcorder and minimize wave perturbation while recording underwater digital images of the seafloor. Color image processing algorithms were applied to estimate the patch coverage area and density, since Anabaena sp. filaments exhibit a characteristic green tone. Results of field tests showed the feasibility of the robotic system to estimate the relative density, distribution, and coverage area of cyanobacteria blooms, preventing the possible impact of direct observation. The robotic system could also be used in surveys of other benthos in the sublittoral zone.

Enhanced decompositions of some cyano complexes during diode array spectrophotometric measurements.

Acidic solutions of the Fe(III), Mo(V), W(V) and Mn(IV) cyano complexes are known to decompose photochemically. These reactions do not take place to an appreciable extent when the cyano complexes are subjected to spectrophotometric measurements by means of conventional spectrophotometers, but they do take place when diode array spectrophotometers are used. The reactions are autoaccelerated, at least in the cases of Mo(V), W(V) and Mn(IV), and consist of photoinduced reductions of the metal ion. This effect should be taken into account in all cases where the measured signal has to be used for quantitative purposes, but its significance must be emphasized mainly in the field of kinetic analysis, where it can lead to large errors. The extent of these reactions, and therefore the size of the errors, can be lowered by using UV cut-off filters and by taking discontinuous measurements with suitable integration times. All these possibilities, which are of general application, are discussed.

Formation and effects of leukotriene B4 in human lymphocytes.

Incubation of human tonsillar B lymphocytes or peripheral blood T lymphocytes with leukotriene (LT) A4 led to the formation of LTB4. Stimulation of these cells with ionophore A23187 did not lead to the synthesis of detectable amounts of leukotrienes. Formation of LTB4 was observed in several monoclonal B- and T-cell lines after incubation with LTA4, but not after stimulation with ionophore A23187. The Burkitt lymphoma cell line Raji was found to possess higher LTA4-hydrolase activity than normal lymphocytes. The expression of the LTA4-hydrolase gene but not the 5-lipoxygenase gene was demonstrated on the transcriptional level in Northern blots and on the translational level by Western blots. Stimulation of human monocytes with ionophore A23187 resulted in the release of LTA4. Coincubations of transformed lymphocytes and monocytes stimulated with ionophore A23187 produced increased amounts of LTB4 as compared with monocytes alone. LTB4 influence on lymphocyte activation was studied and CD23 expression was used as a marker. The expression of this antigen was enhanced on resting B lymphocytes in synergy with B-cell growth-promoting factors. LTB4 also augmented DNA synthesis, cell replication and IgG secretion. These results indicate that extracellular LTA4, released from activated monocytes, is converted by lymphocytes into LTB4 which might cause activation and differentiation of B lymphocytes.

Barrier formation: potential molecular mechanism of enamel fluorosis.

Enamel fluorosis is an irreversible structural enamel defect following exposure to supraoptimal levels of fluoride during amelogenesis. We hypothesized that fluorosis is associated with excess release of protons during formation of hypermineralized lines in the mineralizing enamel matrix. We tested this concept by analyzing fluorotic enamel defects in wild-type mice and mice deficient in anion exchanger-2a,b (Ae2a,b), a transmembrane protein in maturation ameloblasts that exchanges extracellular Cl(-) for bicarbonate. Defects were more pronounced in fluorotic Ae2a,b (-/-) mice than in fluorotic heterozygous or wild-type mice. Phenotypes included a hypermineralized surface, extensive subsurface hypomineralization, and multiple hypermineralized lines in deeper enamel. Mineral content decreased in all fluoride-exposed and Ae2a,b(-/-) mice and was strongly correlated with Cl(-). Exposure of enamel surfaces underlying maturation-stage ameloblasts to pH indicator dyes suggested the presence of diffusion barriers in fluorotic enamel. These results support the concept that fluoride stimulates hypermineralization at the mineralization front. This causes increased release of protons, which ameloblasts respond to by secreting more bicarbonates at the expense of Cl(-) levels in enamel. The fluoride-induced hypermineralized lines may form barriers that impede diffusion of proteins and mineral ions into the subsurface layers, thereby delaying biomineralization and causing retention of enamel matrix proteins.