Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress

Diatoms are unicellular algae that accumulate significant amounts of triacylglycerols as storage lipids when their growth is limited by nutrients. Using biochemical, physiological, bioinformatics, and reverse genetic approaches, we analyzed how the flux of carbon into lipids is influenced by nitrogen stress in a model diatom, Phaeodactylum tricornutum. Our results reveal that the accumulation of lipids is a consequence of remodeling of intermediate metabolism, especially reactions in the tricarboxylic acid and the urea cycles. Specifically, approximately one-half of the cellular proteins are cannibalized; whereas the nitrogen is scavenged by the urea and glutamine synthetase/glutamine 2-oxoglutarate aminotransferase pathways and redirected to the de novo synthesis of nitrogen assimilation machinery, simultaneously, the photobiological flux of carbon and reductants is used to synthesize lipids. To further examine how nitrogen stress triggers the remodeling process, we knocked down the gene encoding for nitrate reductase, a key enzyme required for the assimilation of nitrate. The strain exhibits 40–50% of the mRNA copy numbers, protein content, and enzymatic activity of the wild type, concomitant with a 43% increase in cellular lipid content. We suggest a negative feedback sensor that couples photosynthetic carbon fixation to lipid biosynthesis and is regulated by the nitrogen assimilation pathway. This metabolic feedback enables diatoms to rapidly respond to fluctuations in environmental nitrogen availability.In plants, carbon and nitrogen are directed to specific tissues or structures in accordance with developmental programs. In contrast, unicellular algae flexibly direct carbon and nitrogen to various macromolecules associated with specific intracellular compartments to optimize growth under varying environmental conditions. The signals responsible for this optimization strategy are poorly understood. They clearly are not driven by a developmental program but rather, responses to environmental cues. For example, under optimal growth conditions, ∼40% of the photosynthetically fixed carbon in typical eukaryotic microalga is directed toward the synthesis of amino acids that ultimately are incorporated into proteins (1–3). Over 50 y ago, however, it was recognized that, when nitrogen limits growth, intermediate metabolism is altered, and many microalgae can accumulate storage lipids, mainly in the form of triacylglycerols (TAGs) (4–6). This phenomenon is especially pronounced in diatoms.Diatoms, a highly successful class of eukaryotic algae that rose to ecological prominence during the past 30 My (7), often form massive blooms under turbulent conditions when nutrient supplies are highly variable (8). The ability of these organisms to optimize their growth under such conditions requires coordination of intermediate metabolism of carbon and nitrogen (9, 10). To optimize their growth, the first priority of the cells is to assimilate nitrogen into proteins, which also requires reducing equivalents and carbon skeletons that are primarily supplied by the tricarboxylic acid (TCA) cycle. However, when nitrogen availability decreases, the sink for TCA cycle metabolites declines, and acetyl-CoA, the source of carbon for the cycle, can be shunted toward fatty acid (FA) biosynthesis. Therefore, under nitrogen stress, cellular protein content decreases, whereas storage lipids increase (11, 12). This phenomenon has led to the hypothesis that overexpression of genes involved in lipid biosynthesis may increase the flux of carbon toward lipids (13, 14). Although this phenomenon is well-known, the signals that trigger the process remain unresolved. Genetic manipulations of lipid production in the model diatom, Phaeodactylum tricornutum, are ambiguous. Although there is one report showing that an overexpression of a type II diacylglycerol acyltransferase (DGAT; ProtID 49462) involved in TAG biosynthesis increases the accumulation of natural lipids in P. tricornutum (15), there are several reports indicating that manipulating FA biosynthesis does not significantly affect rates of lipid production (13, 14, 16).Using biochemical, physiological, bioinformatic, and reverse genetic approaches, we examine here how a diatom remodels intermediate metabolism to rapidly respond to nitrogen stress and its resupply. Our results reveal how carbon is redirected toward lipid biosynthesis under nitrogen stress in P. tricornutum.     

In vivo characterization of chronic traumatic encephalopathy using [F-18]FDDNP PET brain imaging

Chronic traumatic encephalopathy (CTE) is an acquired primary tauopathy with a variety of cognitive, behavioral, and motor symptoms linked to cumulative brain damage sustained from single, episodic, or repetitive traumatic brain injury (TBI). No definitive clinical diagnosis for this condition exists. In this work, we used [F-18]FDDNP PET to detect brain patterns of neuropathology distribution in retired professional American football players with suspected CTE (n = 14) and compared results with those of cognitively intact controls (n = 28) and patients with Alzheimer’s dementia (AD) (n = 24), a disease that has been cognitively associated with CTE. [F-18]FDDNP PET imaging results in the retired players suggested the presence of neuropathological patterns consistent with models of concussion wherein brainstem white matter tracts undergo early axonal damage and cumulative axonal injuries along subcortical, limbic, and cortical brain circuitries supporting mood, emotions, and behavior. This deposition pattern is distinctively different from the progressive pattern of neuropathology [paired helical filament (PHF)-tau and amyloid-β] in AD, which typically begins in the medial temporal lobe progressing along the cortical default mode network, with no or minimal involvement of subcortical structures. This particular [F-18]FDDNP PET imaging pattern in cases of suspected CTE also is primarily consistent with PHF-tau distribution observed at autopsy in subjects with a history of mild TBI and autopsy-confirmed diagnosis of CTE.The consensus statement on concussions from the Fourth International Conference on Concussion in Sports (Zurich 2012) (1) defines acute mild traumatic brain injury (mTBI) or cerebral concussion as a brain injury with a complex pathophysiological process induced by biomechanical forces. Cerebral concussion causes white matter axonal injury due to axonal shearing and stretching (2), typically resulting in the rapid onset of short-lived impairment of neurological function that resolves spontaneously and largely reflects a functional disturbance rather than a structural injury. As such, no abnormality is seen on standard structural neuroimaging determinations (1).A number of early literature reports described a neurodegenerative disease associated with a history of repetitive TBI in retired professional boxers (3, 4), with a prevalence rate of up to 47% among retired professional boxers aged 50 y and older who boxed for more than 10 y (5). Initially named “punch drunk syndrome” (3) and dementia pugilistica (4), this syndrome is now known as chronic traumatic encephalopathy (CTE) in the current literature (6, 7).Compelling autopsy evidence (6–8) and neurobehavioral determinations (9) of retired professional American football athletes indicate that a subgroup develops neurodegenerative and clinical changes typical of CTE, a progressive syndrome distinctively different from Alzheimer’s disease (AD), which is the most common form of dementia in the elderly (10). The connection between multiple concussions and subconcussive head impacts (2) and CTE is compelling, because history of repetitive concussions is the strongest risk factor for development of CTE in numerous contact sports (e.g., American football, rugby, boxing, ice hockey, soccer, and professional wrestling), in war veterans with a history of blast or blunt force TBI, and in conditions where trauma to the head occurs for various reasons (e.g., falls during seizures, head-banging in autistic children, motor vehicle and domestic accidents, domestic violence and abuse) (6, 8, 11–14). As with most neurodegenerative diseases, clinical diagnosis remains elusive due to the lack of specificity of CTE clinical symptomatology criteria, and histopathological examination of brain at autopsy is the most definitive diagnostic modality (6, 8, 11).The novel imaging approaches leading to the in vivo characterization of CTE brain neuropathology premortem (e.g., PET) are complementary to structural imaging modalities [e.g., diffusion tensor imaging MRI (DTI MRI)] and offer a specific and sensitive strategy to facilitate diagnosis of CTE. Neuronal and glial fibrillar hyperphosphorylated microtubule-associated protein tau deposits composed of paired helical filament (PHF)-tau are the primary brain proteinopathy of CTE based on autopsy determinations, and their 3R/4R tau isoform ratio is similar to that of AD (11). Their topographically predictable pattern of distribution was used as a basis for a severity staging system of CTE neuropathology (7), ranging from mild (neuropathology stages I and II) to advanced (neuropathology stages III and IV) (7) (Tables S1 and S2). In addition, more than 80% of analyzed pathologically confirmed CTE cases also show transactive response (TAR) DNA-binding protein of ∼43 kDa (TDP-43) either as inclusions in sparse neurites in cortex, medial temporal lobe structures, and brainstem in CTE neuropathology stages I–III, as widespread neuronal and glial inclusions in severe CTE cases (neuropathology stage IV), or in CTE cases with motor neuron disease (7, 15) (Tables S1 and S2). CTE cases also can exhibit the presence of other fibrillar protein aggregates. McKee et al. (7) and Omalu et al. (8) reported that in autopsy determinations, less than half of all CTE cases and less than one third of “pure” CTE cases show amyloid-β (Aβ) deposits, predominantly as scattered cortical diffuse plaques in low density (Tables S1 and S2). Of note is that subjects with Aβ deposits were significantly older than those without. Moreover, their neuropathology was more severe than that in cases without Aβ deposits and was often combined with α-synuclein deposits (7). As an example, as reported by McKee et al. (7), of 30 CTE cases with at least some cortical Aβ deposits (of 68 confirmed CTE cases), 29 brains were from subjects who died in their seventh decade of life and one from a subject who died in his sixth decade.Subsequent to our preliminary report (16), in this work we use [F-18]FDDNP, an imaging agent for fibrillar insoluble protein aggregates (16–20), and PET imaging with the aim of establishing (i) topographic brain localization of [F-18]FDDNP PET signals indicative of fibrillar neuroaggregates in retired professional American football players with suspected CTE (mTBI group) vs. controls (CTRL); (ii) determination of [F-18]FDDNP PET signal patterns in the mTBI group; (iii) presence of [F-18]FDDNP PET signal as a measure of neuropathology in the brain areas involved in mood disorders related neurocircuits; (iv) correlation of [F-18]FDDNP PET results with neuropathology distributions in confirmed CTE cases; (v) differential patterns of [F-18]FDDNP PET signals, and thus deposition of fibrillar neuroaggregates, in the mTBI group with respect to the AD group; and (vi) preliminary demonstration of differences in [F-18]FDDNP PET signal patterns in mTBI cases with different etiology, i.e., contact-sport–related mTBI in retired professional American football players vs. blast-induced mTBI in war veterans. We further intended to demonstrate that tau (vs. Aβ) specificity of high affinity PET molecular imaging probes may not be a necessary requirement when used in CTE subjects with primary proteinopathy in the form of PHF-tau (8): PET imaging probes potentially sensitive to TDP-43 aggregates and Aβ deposits, which are present in higher densities almost exclusively in older CTE cases with more advanced neuropathology (e.g., stage IV), could better define disease progression based on quantification of differences in regional loads of combined neuropathologies because additional neuropathologies appear in predictable topographical and temporal patterns.     

Recent advances in liver transplantation

Advances in liver transplantation continue to evolve but are hampered by continued increasing shortages in donor organs. This has resulted in a high incidence of patients dying while on the United Network for Organ Sharing waiting list. Indeed, we continue to assess ways of expanding the donor pool by using marginal donors, living donor liver transplantation, split liver transplantation, domino transplantation, and hepatic support systems to prolong survival long enough for the patient to undergo liver transplantation. Changes in the liver allocation policy to reduce the number of people dying while waiting for an organ are discussed. Implementation of the model for end-stage liver disease allocation system should help alleviate the problem of increasing deaths of patients while on the waiting list. Recurrent disease, particularly recurrent hepatitis C, continues to be a major problem, and effective therapy is needed to prevent both progression of hepatitis C and recurrence in the graft and avoid retransplantation. The use of pegylated interferon in combination with ribavirin holds promise for improving the success in overcoming recurrent hepatitis C. Finally, advances in immunosuppression have reduced the incidence of acute cellular rejection and chronic rejection. However, these therapies have been fraught with metabolic complications that are now affecting quality of life and long-term survival. Tailoring immunosuppressive regimens to the individual patient is discussed.     

Are there gender differences in left ventricular remodeling after myocardial infarction in rats?

Objective

An unclear issue is whether gender may influence at cardiac remodeling after myocardial infarction (MI). We evaluated left ventricle remodeling in female and male rats post-MI.

Methods

Rats were submitted to anterior descending coronary occlusion. Echocardiographic evaluations were performed on the first and sixth week post-occlusion to determine myocardial infarction size and left ventricle systolic function (FAC, fractional area change). Pulsed Doppler was applied to analyze left ventricle diastolic function using the following parameters: E wave, A wave, E/A ratio. Two-way ANOVA was applied for comparisons, complemented by the Bonferroni test. A P≤=0.05 was considered significant.

Results

There were no significant differences between genders for morphometric parameters on first (MI [Female (FE): 44.0±5.0 vs. Male (MA): 42.0±3.0%]; diastolic [FE: 0.04±0.003 vs. MA: 0.037±0.005, mm/g] and systolic [FE: 0.03±0.0004 vs. MA: 0.028±0.005, mm/g] diameters of left ventricle) and sixth (MI [FE: 44.0±5.0 vs. MA: 42.0±3.0, %]; diastolic [FE: 0.043±0.01 vs. MA: 0.034±0.005, mm/g] and systolic [FE: 0.035±0.01 vs. MA: 0.027±0.005, mm/g] of LV) week. Similar findings were reported for left ventricle functional parameters on first (FAC [FE: 34.0±6.0 vs. MA: 32.0±4.0, %]; wave E [FE: 70.0±18.0 vs. MA: 73.0±14.0, cm/s]; wave A [FE: 20.0±12.0 vs. MA: 28.0±13.0, cm/s]; E/A [FE: 4.9±3.4 vs. MA: 3.3±1.8]) and sixth (FAC [FE: 29.0±7.0 vs. MA: 31.0±7.0, %]; wave E [FE: 85.0±18.0 vs. MA: 87.0±20.0, cm/s]; wave A [FE: 20.0±11.0 vs. MA: 28.0±17.0, cm/s]; E/A [FE: 6.2±4.0 vs. MA: 4.6±3.4]) week.

Conclusion

Gender does not influence left ventricle remodeling post-MI in rats.     

UHMWPE Wear Debris and Tissue Reactions Are Reduced for Contemporary Designs of Lumbar Total Disc Replacements

Background

Lumbar total disc replacement (L-TDR) is a procedure used to relieve back pain and maintain mobility. Contemporary metal-on-polyethylene (MoP) L-TDRs were developed to address wear performance concerns about historical designs, but wear debris generation and periprosthetic tissue reactions for these newer implants have not been determined.

Questions/purposes

The purpose of this study was to determine (1) whether periprosthetic ultrahigh-molecular-weight polyethylene (UHMWPE) wear debris and biological responses were present in tissues from revised contemporary MoP L-TDRs that contain conventional cores fabricated from γ-inert-sterilized UHMWPE; (2) how fixed- versus mobile-bearing design affected UHMWPE wear particle number, shape, and size; and (3) how these wear particle characteristics compare with historical MoP L-TDRs that contain cores fabricated from γ-air-sterilized UHMWPE.

Methods

We evaluated periprosthetic tissues from 11 patients who received eight fixed-bearing ProDisc-L and four mobile-bearing CHARITÉ contemporary L-TDRs with a mean implantation time of 4.1 and 2.7 years, respectively. Histologic analysis of tissues was performed to assess biological responses and polarized light microscopy was used to quantify number and size/shape characteristics of UHMWPE wear particles from the fixed- and mobile-bearing devices. Comparisons were made to previously reported particle data for historical L-TDRs.

Results

Five of seven (71%) fixed-bearing and one of four mobile-bearing L-TDR patient tissues contained at least 4 particles/mm² wear with associated macrophage infiltration. Tissues with wear debris were highly vascularized, whereas those without debris were more necrotic. Given the samples available, the tissue around mobile-bearing L-TDR was observed to contain 87% more, 11% rounder, and 11% less-elongated wear debris compared with tissues around fixed-bearing devices; however, there were no significant differences. Compared with historical L-TDRs, UHMWPE particle number and circularity for contemporary L-TDRs were 99% less (p = 0.003) and 50% rounder (p = 0.003).

Conclusions

In this preliminary study, short-term results suggest there was no significant influence of fixed- or mobile-bearing designs on wear particle characteristics of contemporary L-TDRs, but conventional UHMWPE has notably improved the wear resistance of these devices compared with historical UHMWPE.