Characterization of photodamage to escherichia coli in optical traps

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.     

Transmission potential,skin inflammatory response,and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis

Background

Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector.

Results

The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs).

Conclusion

Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.

     

The genomic organisation and nucleotide sequence of the HLA-SB(DP) alpha gene.

We have isolated a unique fragment of the HLA-DR alpha gene and probed human genomic DNA at low stringency to search for homologous sequences. A minimum of six non-polymorphic cross-hybridizing high molecular weight fragments were found in all DNAs examined. In order to obtain molecular clones of these cross-hybridizing fragments, we constructed lambda and cosmid libraries of human DNA and screened them at low stringency with the HLA-DR alpha gene specific subclone. We have isolated clones corresponding to each of the six fragments and, in this paper, describe those which contain the gene encoding HLA-SB(DP) alpha.     

hESC-Derived Thalamic Organoids Form Reciprocal Projections When Fused with Cortical Organoids

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Anatomical Brain Images Alone Can Accurately Diagnose Chronic Neuropsychiatric Illnesses

Objective

Diagnoses using imaging-based measures alone offer the hope of improving the accuracy of clinical diagnosis, thereby reducing the costs associated with incorrect treatments. Previous attempts to use brain imaging for diagnosis, however, have had only limited success in diagnosing patients who are independent of the samples used to derive the diagnostic algorithms. We aimed to develop a classification algorithm that can accurately diagnose chronic, well-characterized neuropsychiatric illness in single individuals, given the availability of sufficiently precise delineations of brain regions across several neural systems in anatomical MR images of the brain.

Methods

We have developed an automated method to diagnose individuals as having one of various neuropsychiatric illnesses using only anatomical MRI scans. The method employs a semi-supervised learning algorithm that discovers natural groupings of brains based on the spatial patterns of variation in the morphology of the cerebral cortex and other brain regions. We used split-half and leave-one-out cross-validation analyses in large MRI datasets to assess the reproducibility and diagnostic accuracy of those groupings.

Results

In MRI datasets from persons with Attention-Deficit/Hyperactivity Disorder, Schizophrenia, Tourette Syndrome, Bipolar Disorder, or persons at high or low familial risk for Major Depressive Disorder, our method discriminated with high specificity and nearly perfect sensitivity the brains of persons who had one specific neuropsychiatric disorder from the brains of healthy participants and the brains of persons who had a different neuropsychiatric disorder.

Conclusions

Although the classification algorithm presupposes the availability of precisely delineated brain regions, our findings suggest that patterns of morphological variation across brain surfaces, extracted from MRI scans alone, can successfully diagnose the presence of chronic neuropsychiatric disorders. Extensions of these methods are likely to provide biomarkers that will aid in identifying biological subtypes of those disorders, predicting disease course, and individualizing treatments for a wide range of neuropsychiatric illnesses.     

Inositol 1,4,5-trisphosphate receptor ubiquitination is mediated by mammalian Ubc7, a component of the endoplasmic reticulum-associated degradation pathway,and is inhibited by chelation of intracellular Zn2+

In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP3Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome. Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin-protein ligases (E3s). In the present study we have examined the enzymology of ubiquitination of endogenous InsP3Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum-associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p. Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP3R ubiquitination and down-regulation, but that MmUbc6 does not. These data indicate that InsP3Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP3Rs, that are subject to regulated ERAD. Additional studies showed that the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP3R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP3R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process.