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1.
Anal Chem ; 95(37): 13932-13940, 2023 09 19.
Article in English | MEDLINE | ID: mdl-37676066

ABSTRACT

In environmental research, it is critical to understand how toxins impact invertebrate eggs and egg banks, which, due to their tiny size, are very challenging to study by conventional nuclear magnetic resonance (NMR) spectroscopy. Microcoil technology has been extensively utilized to enhance the mass-sensitivity of NMR. In a previous study, 5-axis computer numerical control (CNC) micromilling (shown to be a viable alternative to traditional microcoil production methods) was used to create a prototype copper slotted-tube resonator (STR). Despite the excellent limit of detection (LOD) of the resonator, the quality of the line shape was very poor due to the magnetic susceptibility of the copper resonator itself. This is best solved using magnetic susceptibility-matched materials. In this study, approaches are investigated that improve the susceptibility while retaining the versatility of coil milling. One method involves machining STRs from various copper/aluminum alloys, while the other involves machining ones from an aluminum 2011 alloy and electroplating them with copper. In all cases, combining copper and aluminum to produce resonators resulted in improved line shape and SNR compared to pure copper resonators due to their reduced magnetic susceptibility. However, the copper-plated aluminum resonators showed optimal performance from the devices tested. The enhanced LOD of these STRs allowed for the first 1H-13C heteronuclear multiple quantum coherence (HMQC) of a single intact 13C-labeled Daphnia magna egg (∼4 µg total biomass). This is a key step toward future screening programs that aim to elucidate the toxic processes in aquatic eggs.


Subject(s)
Aluminum , Copper , Animals , Alloys , Biomass , Daphnia
2.
Anal Chem ; 95(38): 14392-14401, 2023 09 26.
Article in English | MEDLINE | ID: mdl-37713676

ABSTRACT

Nuclear magnetic resonance (NMR) is a powerful technique with applications ranging from small molecule structure elucidation to metabolomics studies of living organisms. Typically, solution-state NMR requires a homogeneous liquid, and the whole sample is analyzed as a single entity. While adequate for homogeneous samples, such an approach is limited if the composition varies as would be the case in samples that are naturally heterogeneous or layered. In complex samples such as living organisms, magnetic susceptibility distortions lead to broad 1H line shapes, and thus, the additional spectral dispersion afforded by 2D heteronuclear experiments is often required for metabolite discrimination. Here, a novel, slice-selective 2D, 1H-13C heteronuclear single quantum coherence (HSQC) sequence was developed that exclusively employs shaped pulses such that only spins in the desired volume are perturbed. In turn, this permits multiple volumes in the tube to be studied during a single relaxation delay, increasing sensitivity and throughput. The approach is first demonstrated on standards and then used to isolate specific sample/sensor elements from a microcoil array and finally study slices within a living earthworm, allowing metabolite changes to be discerned with feeding. Overall, slice-selective NMR is demonstrated to have significant potential for the study of layered and other inhomogeneous samples of varying complexity. In particular, its ability to select subelements is an important step toward developing microcoil receive-only arrays to study environmental toxicity in tiny eggs, cells, and neonates, whereas localization in larger living species could help better correlate toxin-induced biochemical responses to the physical localities or organs involved.


Subject(s)
Eggs , Oligochaeta , Humans , Infant, Newborn , Animals , Nuclear Magnetic Resonance, Biomolecular , Hazardous Substances , Metabolomics
3.
Anal Chem ; 94(23): 8523-8532, 2022 06 14.
Article in English | MEDLINE | ID: mdl-35658120

ABSTRACT

Nuclear magnetic resonance (NMR) spectroscopy has played an integral role in medical and environmental metabolic research. However, smaller biological entities, such as eggs and small tissue samples, are becoming increasingly important to better understand toxicity, biological growth/development, and diseases. Unfortunately, their small sizes make them difficult to study using conventional 5 mm NMR probes due to limited sensitivity. The use of microcoil NMR holds great potential for the analysis of such samples, where the coil can be designed to match the sample size to significantly improve NMR mass sensitivity and the filling factor. Here, we compare the potential of planar and Helmholtz microcoil designs to execute complex experiments for the analysis of intact, mass-limited biological samples. The planar coil offers the advantage of an open access design, potentially allowing flow systems to be incorporated and varying sample sizes to be studied; however, its relatively inhomogeneous B1 field leads to reduced NMR performance. The Helmholtz microcoil overcomes this drawback with its symmetrical design, improving B1 homogeneity across the sample but with the caveat that the size and shape of the sample is limited to the spacing between the two parallel coils. The line shape, sensitivity, and RF performance are compared on both coils using standard samples and biological samples. This study found that the Helmholtz microcoil used here considerably outperforms the planar coil in multipulse experiments and has great potential to study complex biological samples in the 50-200 nL range.


Subject(s)
Magnetic Resonance Imaging , Equipment Design , Magnetic Resonance Spectroscopy/methods
4.
Magn Reson Chem ; 60(3): 386-397, 2022 03.
Article in English | MEDLINE | ID: mdl-34647646

ABSTRACT

Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences. Unfortunately, 1 H NMR alone is not very informative for intact biological samples due to line broadening caused by magnetic susceptibility distortions, and 1 H-13 C 2D NMR correlations are required to provide the additional spectral dispersion for metabolic assignments in vivo or in situ. To our knowledge, double-tuned single-sided microcoils have not been applied for the 2D 1 H-13 C analysis of intact 13 C enriched biological samples. Questions include the following: Can 1 H-13 C 2D NMR be performed on single-sided planar microcoils? If so, do they still hold sensitivity advantages over conventional 5 mm NMR technology for mass limited samples? Here, 2D 1 H-13 C HSQC, HMQC, and HETCOR variants were compared and then applied to 13 C enriched broccoli seeds and Daphnia magna (water fleas). Compared to 5 mm NMR probes, the microcoils showed a sixfold improvement in mass sensitivity (albeit only for a small localized region) and allowed for the identification of metabolites in a single intact D. magna for the first time. Single-sided planar microcoils show practical benefit for 1 H-13 C NMR of intact biological samples, if localized information within ~0.7 mm of the 1 mm I.D. planar microcoil surface is of specific interest.


Subject(s)
Daphnia , Magnetic Resonance Imaging , Animals , Magnetic Resonance Spectroscopy/methods , Nuclear Magnetic Resonance, Biomolecular
5.
Neurogenetics ; 16(1): 27-32, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25342198

ABSTRACT

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) represent the most common heritable neuromuscular disorders. Molecular diagnostics of CMT1A/HNPP diseases confirm clinical diagnosis, but their value is limited to the clinical course and prognosis. However, no biomarkers of CMT1A/HNPP have been identified. We decided to explore if the LITAF/SIMPLE gene shared a functional link to the PMP22 gene, whose duplication or deletion results in CMT1A and HNPP, respectively. By studying a large cohort of CMT1A/HNPP-affected patients, we found that the LITAF I92V sequence variant predisposes patients to an earlier age of onset of both the CMT1A and HNPP diseases. Using cell transfection experiments, we showed that the LITAF I92V sequence variant partially mislocalizes to the mitochondria in contrast to wild-type LITAF which localizes to the late endosome/lysosomes and is associated with a tendency for PMP22 to accumulate in the cells. Overall, this study shows that the I92V LITAF sequence variant would be a good candidate for a biomarker in the case of the CMT1A/HNPP disorders.


Subject(s)
Arthrogryposis/genetics , Charcot-Marie-Tooth Disease/genetics , Hereditary Sensory and Motor Neuropathy/genetics , Nuclear Proteins/genetics , Sequence Deletion , Transcription Factors/genetics , Age of Onset , Animals , Arthrogryposis/complications , Arthrogryposis/diagnosis , Arthrogryposis/epidemiology , Biomarkers , Cells, Cultured , Charcot-Marie-Tooth Disease/complications , Charcot-Marie-Tooth Disease/diagnosis , Charcot-Marie-Tooth Disease/epidemiology , Chlorocebus aethiops , Female , Genetic Predisposition to Disease , Hereditary Sensory and Motor Neuropathy/complications , Hereditary Sensory and Motor Neuropathy/diagnosis , Hereditary Sensory and Motor Neuropathy/epidemiology , Humans , Male , Mitochondria/metabolism , Myelin Proteins/metabolism
6.
PLoS One ; 9(7): e103454, 2014.
Article in English | MEDLINE | ID: mdl-25058650

ABSTRACT

Charcot-Marie-Tooth (CMT) disease is one of the most common heritable neuromuscular disorders, affecting 1 in every 2500 people. Mutations in LITAF have been shown to be causative for CMT type 1C disease. In this paper we explore the subcellular localization of wild type LITAF and mutant forms of LITAF known to cause CMT1C (T49M, A111G, G112S, T115N, W116G, L122V and P135T). The results show that LITAF mutants A111G, G112S, W116G, and T115N mislocalize from the late endosome/lysosome to the mitochondria while the mutants T49M, L122V, and P135T show partial mislocalization with a portion of the total protein present in the late endosome/lysosome and the remainder of the protein localized to the mitochondria. This suggests that different mutants of LITAF will produce differing severity of disease. We also explored the effect of the presence of mutant LITAF on wild-type LITAF localization. We showed that in cells heterozygous for LITAF, CMT1C mutants T49M and G112S are dominant since wild-type LITAF localized to the mitochondria when co-transfected with a LITAF mutant. Finally, we demonstrated how LITAF transits to the endosome and mitochondria compartments of the cell. Using Brefeldin A to block ER to Golgi transport we demonstrated that wild type LITAF traffics through the secretory pathway to the late endosome/lysosome while the LITAF mutants transit to the mitochondria independent of the secretory pathway. In addition, we demonstrated that the C-terminus of LITAF is necessary and sufficient for targeting of wild-type LITAF to the late endosome/lysosome and the mutants to the mitochondria. Together these data provide insight into how mutations in LITAF cause CMT1C disease.


Subject(s)
Charcot-Marie-Tooth Disease/genetics , Mitochondria/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Cell Line , Charcot-Marie-Tooth Disease/pathology , Endosomes/metabolism , Humans , Lysosomes/metabolism , Mutation , Nuclear Proteins/chemistry , Protein Transport , Secretory Pathway , Transcription Factors/chemistry
7.
J Virol ; 87(16): 9199-207, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23760249

ABSTRACT

Frog virus 3 (FV3) is the type species of the genus Ranavirus, family Iridoviridae. The genome of FV3 is 105,903 bases in length and encodes 97 open reading frames (ORFs). The FV3 ORF 97R contains a B-cell lymphoma 2 (Bcl-2) homology 1 (BH1) domain and has sequence similarity to the myeloid cell leukemia-1 (Mcl-1) protein, suggesting a potential role in apoptosis. To begin to understand the role of 97R, we characterized 97R through immunofluorescence and mutagenesis. Here we demonstrated that 97R localized to the endoplasmic reticulum (ER) at 24 h posttransfection. However, at 35 h posttransfection, 97R localized to the ER but also began to form concentrated pockets continuous with the nuclear membrane. After 48 h posttransfection, 97R was still localized to the ER, but we began to observe the ER and the outer nuclear membrane invaginating into the nucleus. To further explore 97R targeting to the ER, we created a series of C-terminal transmembrane domain deletion mutants. We found that deletion of 29 amino acids from the C terminus of 97R abolished localization to the ER. In contrast, deletion of 12 amino acids from the C terminus of 97R did not affect 97R localization to the ER. In addition, a hybrid protein containing the 97R C-terminal 33 amino acids was similarly targeted to the ER. These data indicate that the C-terminal 33 amino acids of 97R are necessary and sufficient for ER targeting.


Subject(s)
Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Host-Pathogen Interactions , Ranavirus/physiology , Viral Proteins/metabolism , Virus Replication , Animals , Cell Line , DNA Mutational Analysis , Endoplasmic Reticulum/chemistry , Humans , Microscopy, Fluorescence , Protein Transport , Sequence Deletion
8.
J Virol ; 87(2): 716-23, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23097445

ABSTRACT

Iridoviruses are a family of large double-stranded DNA (dsDNA) viruses that are composed of 5 genera, including the Lymphocystivirus, Ranavirus, Megalocytivirus, Iridovirus, and Chloriridovirus genera. The frog virus 3 (FV3) 75L gene is a nonessential gene that is highly conserved throughout the members of the Ranavirus genus but is not found in other iridoviruses. FV3 75L shows high sequence similarity to a conserved domain found in the C terminus of LITAF, a small cellular protein with unknown function. Here we show that FV3 75L localizes to early endosomes, while LITAF localizes to late endosomes/lysosomes. Interestingly, when FV3 75L and LITAF are cotransfected into cells, LITAF can alter the subcellular localization of FV3 75L to late endosomes/lysosomes, where FV3 75L then colocalizes with LITAF. In addition, we demonstrated that virally produced 75L colocalizes with LITAF. We confirmed a physical interaction between LITAF and FV3 75L but found that this interaction was not mediated by two PPXY motifs in the N terminus of LITAF. Mutation of two PPXY motifs in LITAF did not affect the colocalization of LITAF and FV3 75L but did change the location of the two proteins from late endosomes/lysosomes to early endosomes.


Subject(s)
Endosomes/metabolism , Host-Pathogen Interactions , Protein Interaction Mapping , Ranavirus/pathogenicity , Viral Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Humans , Molecular Sequence Data , Nuclear Proteins , Protein Binding , Protein Transport , Ranavirus/physiology , Sequence Homology, Amino Acid , Transcription Factors
9.
PLoS One ; 7(1): e30003, 2012.
Article in English | MEDLINE | ID: mdl-22276139

ABSTRACT

LITAF is a 161 amino acid cellular protein which includes a proline rich N-terminus and a conserved C-terminal domain known as the simple-like domain. Mutations in LITAF have been identified in Charcot-Marie tooth disease, a disease characterized by protein aggregates. Cells transfected with cellular LITAF reveal that LITAF is localized to late endosomes/lysosomes. Here we investigated the intracellular localization of endogenous LITAF. We demonstrated that endogenous LITAF accumulates at a discrete cytoplasmic site in BGMK cells that we identify as the aggresome. To determine the domain within LITAF that is responsible for the localization of LITAF to aggresomes, we created a construct that contained the C-terminal simple-like domain of LITAF and found that this construct also localizes to aggresomes. These data suggest the simple-like domain is responsible for targeting endogenous LITAF to the aggresome.


Subject(s)
Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Cell Line , Charcot-Marie-Tooth Disease/metabolism , Chlorocebus aethiops , DNA-Binding Proteins , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Fluorescent Antibody Technique , Humans , Mice , Multiprotein Complexes/genetics , Mutation , Nedd4 Ubiquitin Protein Ligases , Nuclear Proteins/genetics , Polymerase Chain Reaction , Transcription Factors/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
10.
Einstein (Säo Paulo) ; 8(1)jan.-mar. 2010. tab, graf
Article in Portuguese | LILACS | ID: lil-542628

ABSTRACT

Objective: To compare the profile of women looking for gynecological care to the profile of women invited to participate in the program, assessing breast and cervical cancer risk factors in each group and comparing Papanicolaous test and mammography results. Methods: Medical records of 46 women participating in a breast and cervical cancer prevention program and 42 medical reports of women that regularly visited the primary healthcare unit from August to December 2006 were examined. Results: The mean interval between the last Papanicolaous tests was of approximately 19.7 months when comparing women visiting their physician and 25.3 participants in the program. There was one case (1.1%) of high grade intraepithelial lesion in one woman included in the program. Regarding breast cancer, when comparing both groups, we verified that all women above the age of 40 years that participated in the program underwent mammography; this was not verified in the group seeing a physician. This shows the efficacy of this screening, actively looking for women in the age group at risk for breast cancer. Conclusions: Active search is important to recruit women; the screening program needs improvement to show its real impact on morbidity and mortality of these cancers.


Objetivo: Comparar o perfil das mulheres que buscam atendimento ginecológico ao perfil de mulheres convidadas a participarem do programa, avaliando-se fatores de risco para câncer de mama e de colo uterino presentes em cada grupo e comparando os resultados dos exames de Papanicolaou e mamografia. Métodos: Foi realizado levantamento de 46 prontuários de mulheres de um programa de prevenção de câncer de colo do útero e mama e de 42 prontuários de pacientes que consultaram rotineiramente o médico da unidade básica de saúde entre agosto e dezembro de 2006. Resultados: Constatou-se que o intervalo médio entre os dois últimos exames de Papanicolaou foi de aproximadamente 19,7 meses entre as mulheres que consultaram o médico e 25,3 entre as participantes do programa. Houve um caso (1,1%) de lesão intraepitelial de alto grau em uma paciente incluída no programa. Com relação ao câncer de mama, comparando-se os dois grupos, observou-se que todas as mulheres acima de 40 anos do Grupo Programa realizaram mamografia, o que não ocorreu no Grupo Médico. Isso mostra a eficiência do programa de rastreamento na busca ativa de mulheres que se encontram na faixa etária de risco para o câncer de mama. Conclusões: A busca ativa é importante para o recrutamento de mulheres, havendo necessidade de aprimorar o programa de rastreamento para evidenciar seu impacto na morbiletalidade das doenças.

11.
Einstein (Sao Paulo) ; 8(1): 34-9, 2010 Mar.
Article in English, Portuguese | MEDLINE | ID: mdl-26761750

ABSTRACT

OBJECTIVE: To compare the profile of women looking for gynecological care to the profile of women invited to participate in the program, assessing breast and cervical cancer risk factors in each group and comparing Papanicolaou's test and mammography results. METHODS: Medical records of 46 women participating in a breast and cervical cancer prevention program and 42 medical reports of women that regularly visited the primary healthcare unit from August to December 2006 were examined. RESULTS: The mean interval between the last Papanicolaou's tests was of approximately 19.7 months when comparing women visiting their physician and 25.3 participants in the program. There was one case (1.1%) of high grade intraepithelial lesion in one woman included in the program. Regarding breast cancer, when comparing both groups, we verified that all women above the age of 40 years that participated in the program underwent mammography; this was not verified in the group seeing a physician. This shows the efficacy of this screening, actively looking for women in the age group at risk for breast cancer. CONCLUSIONS: Active search is important to recruit women; the screening program needs improvement to show its real impact on morbidity and mortality of these cancers.

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