Effect of C-Terminal Protein Tags on Pentitol and l-Arabinose Transport by Ambrosiozyma monospora Lat1 and Lat2 Transporters in Saccharomyces cerevisiae

Functional expression in heterologous hosts is often less successful for integral membrane proteins than for soluble proteins. Here, two Ambrosiozyma monospora transporters were successfully expressed in Saccharomyces cerevisiae as tagged proteins. Growth of A. monospora on l-arabinose instead of glucose caused transport activities of l-arabinose, l-arabitol, and ribitol, measured using l-[1-³H]arabinose, l-[¹⁴C]arabitol, and [¹⁴C]ribitol of demonstrated purity. A. monospora LAT1 and LAT2 genes were cloned earlier by using their ability to improve the growth of genetically engineered Saccharomyces cerevisiae on l-arabinose. However, the l-arabinose and pentitol transport activities of S. cerevisiae carrying LAT1 or LAT2 are only slightly greater than those of control strains. S. cerevisiae carrying the LAT1 or LAT2 gene fused in frame to the genes for green fluorescent protein (GFP) or red fluorescent protein (mCherry) or adenylate kinase (AK) exhibited large (>3-fold for LAT1; >20-fold for LAT2) increases in transport activities. Lat1-mCherry transported l-arabinose with high affinity (K_m ≈ 0.03 mM) and l-arabitol and ribitol with very low affinity (K_m ≥ 75 mM). The Lat2-GFP, Lat2-mCherry, and Lat2-AK fusion proteins could not transport l-arabinose but were high-affinity pentitol transporters (K_ms ≈ 0.2 mM). The l-arabinose and pentitol transport activities of A. monospora could not be completely explained by any combination of the observed properties of tagged Lat1 and Lat2, suggesting either that tagging and expression in a foreign membrane alters the transport kinetics of Lat1 and/or Lat2 or that A. monospora contains at least one more l-arabinose transporter.     

Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.     

Cerebral Cortex Hyperthyroidism of Newborn Mct8-Deficient Mice Transiently Suppressed by Lat2 Inactivation

Thyroid hormone entry into cells is facilitated by transmembrane transporters. Mutations of the specific thyroid hormone transporter, MCT8 (Monocarboxylate Transporter 8, SLC16A2) cause an X-linked syndrome of profound neurological impairment and altered thyroid function known as the Allan-Herndon-Dudley syndrome. MCT8 deficiency presumably results in failure of thyroid hormone to reach the neural target cells in adequate amounts to sustain normal brain development. However during the perinatal period the absence of Mct8 in mice induces a state of cerebral cortex hyperthyroidism, indicating increased brain access and/or retention of thyroid hormone. The contribution of other transporters to thyroid hormone metabolism and action, especially in the context of MCT8 deficiency is not clear. We have analyzed the role of the heterodimeric aminoacid transporter Lat2 (Slc7a8), in the presence or absence of Mct8, on thyroid hormone concentrations and on expression of thyroid hormone-dependent cerebral cortex genes. To this end we generated Lat2^-/-, and Mct8^-/yLat2 ^-/- mice, to compare with wild type and Mct8^-/y mice during postnatal development. As described previously the single Mct8 KO neonates had a transient increase of 3,5,3′-triiodothyronine concentration and expression of thyroid hormone target genes in the cerebral cortex. Strikingly the absence of Lat2 in the double Mct8Lat2 KO prevented the effect of Mct8 inactivation in newborns. The Lat2 effect was not observed from postnatal day 5 onwards. On postnatal day 21 the Mct8 KO displayed the typical pattern of thyroid hormone concentrations in plasma, decreased cortex 3,5,3′-triiodothyronine concentration and Hr expression, and concomitant Lat2 inactivation produced little to no modifications. As Lat2 is expressed in neurons and in the choroid plexus, the results support a role for Lat2 in the supply of thyroid hormone to the cerebral cortex during early postnatal development.     

A comparative study in Porcellio laevis (Lat.) and Armadillidium vulgare (Lat.): response to temperature, photoperiod and oxygen concentration.

Relationship of body weight, imposed fasting, temperature, light intensity, and oxygen concentration to oxygen consumption in Porcellio laevis and Armadillidium vulgare has been investigated in a series of laboratory experiments. It was observed that (1) the metabolic response in the two species to temperature change was a uniform increase of oxygen consumption with increasing temperature from 15 degrees C to 60 degrees C. Beyond 30 degrees C, the oxygen consumption in each species fell, and the thermal death point was reached at about 40 degrees C. (2) The response to decreasing oxygen concentrations was a corresponding decrease in oxygen consumption. Armadillidium vulgare was a partial regulator while Porcellio laevis was able to conform its internal state to the changing oxygen levels. (3) In each species there was a decrease in metabolic rate with increasing body weight. (4) On the basis of their general activity level and oxygen consumption rate, Porcellio appeared to be a nocturnal species, while Armadillidium had a day active metabolism.     

The determination of the antagonistic activity of Solco lactobacteria (Lactobacillus acidophilus Lat 11/83) using gnotobiotic technology

Lactobacillus acidophilus strain Lat 11/83 has been used for the study of its antagonistic activity with respect to pathogenic microorganisms in experiments on conventional germ-free animals. The results of these experiments indicate that the above strain may be recommended as a highly active antagonist for the treatment and prophylaxis of intestinal dysbacteriosis of different etiology.     

The Crime Lab Project

The Crime Lab Project takes an economical, hands-on, interdisciplinary approach to studying the career of forensics in the middle or high school classroom. The project requires a week of class time, uses common household items, can be applied to students with different levels of ability, and is considered durable. In addition, the project covers topics that address national science standards such as career development, the scientific method, lab procedures, teamwork, and scientific literary skills. Students also learn to distinguish between observations and conclusions.     

Itk/Emt/Tsk activation in response to CD3 cross-linking in Jurkat T cells requires ZAP-70 and Lat and is independent of membrane recruitment.

The Tec family tyrosine kinase, Itk has been implicated in T cell antigen receptor (TCR) signaling, yet little is known about Itk regulation. Here, we investigate the role of the tyrosine kinase ZAP-70 in regulating Itk. Whereas Itk was activated in Jurkat T cells in response to CD3 cross-linking, Itk activation was defective in the ZAP-70-deficient P116 Jurkat T cell line. Itk responsiveness to TCR engagement was restored in P116 cells stably transfected with ZAP-70 cDNA. ZAP-70 itself could not directly phosphorylate the Itk kinase domain, indicating an indirect regulation of Itk activity. No role was found for ZAP-70 in regulating Itk recruitment to the plasma membrane, an event that has been suggested to be rate-limiting for the activation of Tec family kinases. Indeed, Itk was found to be constitutively targeted to the membrane fraction in both Jurkat and P116 cells. Lat, a prominent in vivo substrate of ZAP-70 that mediates assembly of multimolecular signaling complexes at the plasma membrane of T cells was also found to be required for TCR-stimulated Itk activation. Itk could not be activated by CD3 cross-linking in a Lat-negative cell line, unless Lat expression was restored. Lat and Itk were observed to co-associate in response to CD3 cross-linking in Jurkat T cells, but not in P116 T cells. The Lat-Itk association correlated with Lat tyrosine phosphorylation, which was deficient in the P116 T cells. These data suggest that ZAP-70 and Lat play important, probably sequential, roles in regulating the activation of Itk following TCR engagement.     

The Cardiac Electrophysiology Web Lab

Computational modeling of cardiac cellular electrophysiology has a long history, and many models are now available for different species, cell types, and experimental preparations. This success brings with it a challenge: how do we assess and compare the underlying hypotheses and emergent behaviors so that we can choose a model as a suitable basis for a new study or to characterize how a particular model behaves in different scenarios? We have created an online resource for the characterization and comparison of electrophysiological cell models in a wide range of experimental scenarios. The details of the mathematical model (quantitative assumptions and hypotheses formulated as ordinary differential equations) are separated from the experimental protocol being simulated. Each model and protocol is then encoded in computer-readable formats. A simulation tool runs virtual experiments on models encoded in CellML, and a website (https://chaste.cs.ox.ac.uk/WebLab) provides a friendly interface, allowing users to store and compare results. The system currently contains a sample of 36 models and 23 protocols, including current-voltage curve generation, action potential properties under steady pacing at different rates, restitution properties, block of particular channels, and hypo-/hyperkalemia. This resource is publicly available, open source, and free, and we invite the community to use it and become involved in future developments. Investigators interested in comparing competing hypotheses using models can make a more informed decision, and those developing new models can upload them for easy evaluation under the existing protocols, and even add their own protocols.     

Coelomycete Fungi in the Clinical Lab

Most human mycoses are incited by fungi that replicate by the production of asexual propagules (usually called conidia), which are readily dispersed throughout nature and also in vitro. Fungi that are acquired in this manner include a diversity of yeasts and dermatophytes, as well as those whose acquisition is primarily pulmonary, such as the thermally dimorphic primary pathogens, and several opportunistic genera such as Aspergillus, Fusarium and others. Recent reports, however, indicate an increasing number of infections, ranging from superficial to systemic, that are due to coelomycetous fungi. In these fungi, the asexual spores are produced within fruiting bodies referred to as a conidiomata. As these conidia are less frequently airborne, the method of acquisition is commonly by traumatic implantation of plant material or by a diversity of contaminated fomites, rather than by inhalation. These infections are also more commonly seen in immunocompromised individuals. In this work, we have attempted to provide a historial overview as well as the current taxonomy for this group of fungi, an up-to-date assessment of the scope of infections caused by various coelomycetous genera, their clinical entities, useful tools for isolation and identification, and general guidelines for the appropriate management and treatment of these mycoses.     

Next-Generation Sequencing in the Mycology Lab

New state-of-the-art techniques in sequencing offer valuable tools in both detection of mycobiota and in understanding of the molecular mechanisms of resistance against antifungal compounds and virulence. Introduction of new sequencing platform with enhanced capacity and a reduction in costs for sequence analysis provides a potential powerful tool in mycological diagnosis and research. In this review, we summarize the applications of next-generation sequencing techniques in mycology.     

Occurrence and morphometry of the hypopharyngeal glands inScaptotrigona postica Lat. (Hymenoptera, Apidae, Meliponinae)

As compared toApis mellifera where only workers have hypopharyngeal glands, inScaptotrigona postica, these glands occur in workers, queens and males. They are composed of two long axial ducts with many unicellular secretory alveoli interconnected by secretory canaliculi. The axial ducts are longer in males than in workers, but the alveolar areas of queens and males are generally smaller. In workers the alveoli have their greatest size in the nurses or middle-aged individuals while in queens and males they are larger in newly emerged individuals. The results indicate that the glands in workers may produce food for the brood as inA. mellifera, since they are well developed in the nurse workers. However, the function of the glands in queens and males remains to be clarified since these individuals have no part in brood care.     

Fungal Biofilms in the Clinical Lab Setting

Device-related infections are often associated with biofilms (microbial communities encased within polysaccharide-rich extracellular matrix) formed by pathogens on surfaces of these devices. Candida species are the most common fungi isolated from infections associated with catheters and dentures, and both Candida and Fusarium are commonly isolated from contact lens–related infections such as fungal keratitis. These biofilms exhibit decreased susceptibility to most antimicrobial agents, which contributes to the persistence of infection. Drug resistance in fungal biofilms is multifactorial and phase-dependent; for example, efflux pumps mediate resistance in biofilms during early phase, whereas altered membrane sterol composition contributes to resistance in mature phase. Both substrate type and surface coatings play an important role in the pathogenesis of device-related fungal biofilms. Host immune cells influence the ability of Candida to form biofilms in vitro. This review summarizes recent advances in research on fungal biofilms and discusses their clinical relevance.