Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids.

Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family is increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically-engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach strengthens our ability to quantify and characterize the inherent diversity of acyl chains across the lipidome.

C9orf72 poly(PR) mediated neurodegeneration is associated with nucleolar stress.

The ALS/FTD-linked intronic hexanucleotide repeat expansion in the C9orf72 gene is aberrantly translated in the sense and antisense directions into dipeptide repeat proteins, among which poly proline-arginine (PR) displays the most aggressive neurotoxicity in-vitro and in-vivo. PR partitions to the nucleus when heterologously expressed in neurons and other cell types. We show that by lessening the nuclear accumulation of PR, we can drastically reduce its neurotoxicity. PR strongly accumulates in the nucleolus, a nuclear structure critical in regulating the cell stress response. We determined that, in neurons, PR caused nucleolar stress and increased levels of the transcription factor p53. Downregulating p53 levels also prevented PR-mediated neurotoxicity both in in-vitro and in-vivo models. We investigated if PR could induce the senescence phenotype in neurons. However, we did not observe any indications of such an effect. Instead, we found evidence for the induction of programmed cell death via caspase-3 activation.

Glucose Hypometabolism Prompts RAN Translation and Exacerbates C9orf72-related ALS/FTD Phenotypes.

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, although its potential role in disease pathogenesis is unknown. Here, we identified alterations in glucose metabolic pathways and ATP levels in the brain of asymptomatic C9-BAC mice. We found that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also found that one of the arginine-rich DPRs (PR) can directly contribute to glucose metabolism and metabolic stress. These findings provide a mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and support a feedforward loop model that opens several opportunities for therapeutic intervention.

C9orf72 poly(PR) mediated neurodegeneration is associated with nucleolar stress.

The ALS/FTD-linked intronic hexanucleotide repeat expansion in the C9orf72 gene is translated into dipeptide repeat proteins, among which poly-proline-arginine (PR) displays the most aggressive neurotoxicity in-vitro and in-vivo . PR partitions to the nucleus when expressed in neurons and other cell types. Using drosophila and primary rat cortical neurons as model systems, we show that by lessening the nuclear accumulation of PR, we can drastically reduce its neurotoxicity. PR accumulates in the nucleolus, a site of ribosome biogenesis that regulates the cell stress response. We examined the effect of nucleolar PR accumulation and its impact on nucleolar function and determined that PR caused nucleolar stress and increased levels of the transcription factor p53. Downregulating p53 levels, either genetically or by increasing its degradation, also prevented PR-mediated neurotoxic phenotypes both in in-vitro and in-vivo models. We also investigated whether PR could cause the senescence phenotype in neurons but observed none. Instead, we found induction of apoptosis via caspase-3 activation. In summary, we uncovered the central role of nucleolar dysfunction upon PR expression in the context of C9-ALS/FTD.

Sagittal plane analysis of head, neck, and trunk kinematics and electromyographic activity during locomotion.

STUDY DESIGN: Descriptive study examining kinematic and electromyographic (EMG) patterns of the upper body during walking. OBJECTIVE: To examine trunk, neck, and head movements to determine a mechanism for upper body stabilization during walking. BACKGROUND: Dynamic balance of the upper body during walking provides a stable base for function of sensory systems. Prior investigations of upper body motion during walking were limited to examination of isolated segments, or examination of the upper body as a single unit. In our study, the upper body is examined as 3 segments: the trunk, neck, and head. METHODS AND MEASURES: Sagittal plane walking patterns were examined in 8 unimpaired young adults. Markers placed on the trunk, neck, and head segments were recorded on videotape. Angles were calculated with respect to an external horizontal reference to determine segment position relative to space. EMG measures were obtained from erector spinae, rectus abdominus, semispinalis capitis, and sternocleidomastoid muscles. RESULTS: Results showed dynamic stability was accomplished through maintenance of a posture where the trunk was flexed, the neck was extended and the head was flexed. The trunk segment demonstrated greatest stability with the neck being the least stable of the 3 segments. Movements of upper body segments showed a tendency for the head and neck to move opposite to the trunk. EMG data demonstrated erector spinae muscle activity occurring near heel contact of each limb followed by trunk extension. The remaining muscles exhibited variable patterns of activity. CONCLUSIONS: These data indicate that movements of the upper body help to maintain a posture that promotes stability of these segments during walking. The trunk was the most stable of the three segments thereby, providing a stable platform for head and neck movement. Erector spinae muscle activity contributed to upper body movements by extending the trunk to maintain balance at heel contact. These results provide a basis for studying changes in dynamic stability that occur with age.

Lymphoid markers, activation markers, and adhesion molecules in cutaneous biopsy specimens from HIV+ patients with disease progression. The Military Medical Consortium for the Advancement of Retroviral Research.

BACKGROUND: One important factor in understanding the pathogenesis of human immune deficiency virus (HIV) disease is documenting the patterns of immune dysregulation present in HIV-positive patients. The cells which home to skin are mainly certain subsets of T cells and, as opposed to the peripheral blood, where circulating factors may inhibit terminal phenotypic differentiation, the cutaneous environment potentiates differentiation during cutaneous eruptions. OBJECTIVE: The authors' aim was to characterize the inflammatory dermatoses in biopsy specimens from HIV-positive patients with immunohistochemical stains for lymphoid markers, activation markers, and adhesion molecules and to determine if there was any correlation with the type of dermatosis and the HIV-disease stage. METHODS: Lymphoid and activation markers as well as adhesion molecules were studied on cutaneous biopsy specimens from 96 inflammatory dermatoses in HIV-positive patients. The dermatoses included psoriasiform dermatoses with and without a lichenoid component, perivascular lymphoid dermatoses, perivascular and periadnexal inflammatory dermatoses, spongiotic dermatoses, granulomatous dermatoses, and neutrophilic dermatoses with and without vasculitis. RESULTS: Although there was a decrease in CD4/CD8 ratios in the cutaneous inflammatory dermatoses with progression of the disease, the ratios of CD4/CD8 cells were far higher than those in the peripheral blood. There were also increasing numbers of CD23+ cells and increased E-Selectin expression on endothelial cells from the early stages of disease, with no consistent pattern of ICAM-1 expression on epithelial cells with disease progression. CONCLUSIONS: The expression of lymphoid markers, activation markers, and adhesion molecules in the skin with progression of HIV disease, is consistent with a T helper (Th)1 to Th0/Th2 cytokine pattern of immune dysregulation. This cytokine pattern may be modified by the cytopathic effects of HIV on lymphoid and dendritic populations and by effects of other concurrent infections. Significant numbers of CD4+ T cells in skin infiltrates, with low peripheral CD4 T-cell counts, suggest that the cutaneous T-cell populations may be distinctive.

Percentage of reversibly and irreversibly sickled cells are altered by the method of blood drawing and storage conditions.

We previously reported that the percentage of reversibly and irreversibly sickled cells (RSC and ISC, respectively) in the blood of patients with sickle cell disease is strongly influenced by the method of blood drawing (PNAS 91:12589, 1994). We now document the effect of blood storage conditions on the percentage of RSC and ISC. The percentage of RSC was lowest when blood was stored at 0 degree C, while the percentage of RSC was highest in specimens kept at 37 degrees C. At room temperature, the percentage of RSC increased slightly over 8 hours. The percentage of ISC was also temperature dependent and was reduced significantly upon cooling. Our results showed that many ISC reverted to a discoidal shape after 3 hrs of cooling after treatment of blood with oxygen or carbon monoxide. Since no Hb S polymers were detected in ISC treated with oxygen or carbon monoxide, the time required for shape restoration may be attributed to the membrane. We measured ISC levels of 10 patients with consideration of storage temperature and compared the values with those determined by the conventional method and also with those published previously.

Mitogen-activated lymphocytes of normals and glioma patients produce factors with anti-glioblastoma activity in vitro.

Human glioblastoma cell lines showed profound suppression of both DNA and RNA synthesis when exposed to supernatants (SNs) of mitogen-activated blood mononuclear cells. Cloning efficiency of these glioma cells also decreased 10- to 500-fold. In monolayer cultures, growth inhibition was evident within 12 h of adding SN and peaked at 24 h. A decrease in absolute cell number was evident by 72 h. The inhibitory effect of SNs, however, was not permanent as more cells entered S-phase when SN-treated cultures were refed with fresh medium (without SN). The factor(s) responsible for this inhibitory activity was a product of lymphocytes and was produced in comparable amounts by cells of normal blood donors and patients with glioma. The compromised immunological status of glioblastoma patients did not influence their capacity to produce cytostatic lymphokines.

Residual anterior pituitary function following transsphenoidal resection of pituitary macroadenomas.

A series of 84 patients with pituitary adenomas greater than 1 cm in diameter is presented. Full preoperative and postoperative endocrine evaluations were carried out, and the effects of transsphenoidal surgery on remaining anterior pituitary function were analyzed. Of the patients who had normal anterior pituitary function before surgery, 78% retained normal function after surgery. Thirty-three percent of those patients with pituitary deficits who did not have panhypopituitarism before surgery had improved function after surgery; 33% had worsened function after surgery. None of the patients with panhypopituitarism before surgery regained function after surgery. Transsphenoidal surgery carries an acceptable risk for sacrificing anterior pituitary function, but the risk is greater in patients with larger tumors and preoperatively compromised pituitary function.