Ultrastructural examination of cryodamage in Paracentrotus lividus eggs during cryopreservation.

This study examinates the challenges of cryopreserving sea urchin (Paracentrotus lividus) eggs, a task hindered by factors like low membrane permeability and high sensitivity to cryoprotective agents (CPAs). While successful cryopreservation has been achieved for some marine invertebrates, eggs remain problematic due to their unique characteristics. The study explores the impact of various CPAs and cryopreservation techniques on sea urchin eggs, employing scanning and transmission electron microscopy to analyze cellular damage. The findings reveal that exposure to low CPA concentrations (0.5 M) did not induce significant damage to eggs. However, high concentrations (3 M) proved highly detrimental. Every cryopreservation approach investigated in this study resulted in irreversible damage to the sea urchin eggs, rendering them nonviable for future use. The research sheds light on the importance of understanding the structural alterations induced by CPAs and cryopreservation methods. This knowledge is essential for refining cryopreservation methods, potentially paving the way for successful preservation of these challenging cells.

Longitudinal progression of blood biomarkers reveals a key role of astrocyte reactivity in preclinical Alzheimer's disease.

Defining the progression of blood biomarkers of Alzheimer's disease (AD) is essential for targeting treatments in patients most likely to benefit from early intervention. We delineated the temporal ordering of blood biomarkers a decade prior to the onset of AD symptoms in participants in the Baltimore Longitudinal Study of Aging. We show that increased astrocyte reactivity, assessed by elevated glial fibrillary acidic protein (GFAP) levels is an early event in the progression of blood biomarker changes in preclinical AD. In AD-converters who are initially cognitively unimpaired (N=158, 377 serial plasma samples), higher plasma GFAP levels are observed as early as 10-years prior to the onset of cognitive impairment due to incident AD compared to individuals who remain cognitively unimpaired (CU, N=160, 379 serial plasma samples). Plasma GFAP levels in AD-converters remain elevated 5-years prior to and coincident with the onset of cognitive impairment due to AD. In participants with neuropathologically confirmed AD, plasma GFAP levels are elevated relative to cognitively normal individuals and intermediate in those who remain cognitively unimpaired despite significant AD pathology (asymptomatic AD). Higher plasma GFAP levels at death are associated with greater severity of both neuritic plaques and neurofibrillary tangles. In the 5XFAD transgenic model of AD, we observed greater GFAP levels in the cortex and hippocampus of transgenic mice relative to wild-type prior to the development of cognitive impairment. Reactive astrocytosis, an established biological response to neuronal injury, may be an early initiator of AD pathogenesis and a promising therapeutic target.

Exploring the feasibility of cryopreserving larvae of the common cockle (Cerastoderma edule) for hatchery production.

The decline of natural populations of the common cockle (Cerastoderma edule) through the European coast is posing a threat to local small-scale fisheries. These declines are primarily attributed to the prevalence of several pathogens and the disseminated neoplasia in cockle populations. The institution of a biobank of cryopreserved larvae could enhance hatchery production and help the restocking. The present work aimed at the development of a cryopreservation protocol for larvae of the common cockle using the mollusk cryopreservation protocols designed in our laboratory. Toxicity bioassays and short-term cryopreservation experiments were performed for protocol optimization according with cellular tolerance. Once settled, the viability of cryopreserved larvae was studied long term. Toxicity tests evidenced high tolerance of larvae against detrimental effects of Cryoprotecting Agents (CPAs). Cryopreservation of 48 h-old D-larva showed a 100% survival when increasing the equilibrium time from 15 to 60 min and using Propylene-Glycol (PG) + 0.4 M Trehalose (TRE) in Filtered Sea Water (FSW) and 60 min of exposure to CPA solution before slow-cooling. However, when cryopreserving the older larvae, the variation in equilibrium times hardly showed any effect but 10% Ethylene-Glycol (EG) + 0.4 M TRE and 60 min of exposure yielded the best relative survivorship (100%). Cryopreservation caused a significant delay on the growth rate of the latest larval stage. However, cryopreserved larvae survived to day 4-6, while 30 ± 12.17% of control larvae developed into pediveliger stage, of which 50% settled and transformed into juvenile cockles. These results demonstrated the role of the cell-type specificity in cryopreservation and highlight the importance of studying potential long-term effects of this tool to ensure the viability of the protocols.

Long-term study on survival and development of successive generations of Mytilus galloprovincialis cryopreserved larvae.

Shellfish aquaculture needs the development of new tools for the improvement of good practices avoiding the reliance on natural spat collection to increase production efficiently. The aim of this work was to improve the cryopreservation protocol for Mytilus galloprovincialis larvae described in Paredes et al. (in: Wolkers, Oldenhof (eds) Cryopreservation and freeze-drying protocol, methods in molecular biology, Humana Press, 2021, pp 2180, https://doi.org/10.1007/978-1-0716-0783-1_18 ). Moreover, the capability of producing adult mussels from cryopreserved 72 h-old D-larvae and potential long-term effects of cryopreservation through progenies were evaluated. The selection of 72-h old D-larvae for cryopreservation yielded 75% of recovery, higher than 50% from trochophores. The best combination was 10% Ethylene-Glycol + 0.4 M Trehalose in Filtered Sea Water (FSW) with cooling at - 1 °C/min and a water bath at 35 °C for thawing. Sucrose (SUC) solutions did not improve larval recovery (p > 0.05). At settlement, 5.26% of cryopreserved F1 larvae survived and over 70% settled. F2 cryopreservation produced 0.15% survival of spat and settlement varied from 35 to 50%. The delay of shell size showed on cryopreserved larvae declined throughout larval rearing without significant differences with controls from settlement point (p > 0.05). Long-term experiments showed that it is possible to obtain adult mussels from cryopreserved larvae and this tool does not compromise the quality of following progenies, neither for cryopreservation nor post-thawing development of them.

Maximum in density of electrolyte solutions: Learning about ion-water interactions and testing the Madrid-2019 force field.

In this work, we studied the effect of Li+, Na+, K+, Mg2+, and Ca2+ chlorides and sulfates on the temperature of maximum density (TMD) of aqueous solutions at room pressure. Experiments at 1 molal salt concentration were carried out to determine the TMD of these solutions. We also performed molecular dynamics simulations to estimate the TMD at 1 and 2 m with the Madrid-2019 force field, which uses the TIP4P/2005 water model and scaled charges for the ions, finding an excellent agreement between experiment and simulation. All the salts studied in this work shift the TMD of the solution to lower temperatures and flatten the density vs temperature curves (when compared to pure water) with increasing salt concentration. The shift in the TMD depends strongly on the nature of the electrolyte. In order to explore this dependence, we have evaluated the contribution of each ion to the shift in the TMD concluding that Na+, Ca2+, and SO4 2- seem to induce the largest changes among the studied ions. The volume of the system has been analyzed for salts with the same anion and different cations. These curves provide insight into the effect of different ions upon the structure of water. We claim that the TMD of electrolyte solutions entails interesting physics regarding ion-water and water-water interactions and should, therefore, be considered as a test property when developing force fields for electrolytes. This matter has been rather unnoticed for almost a century now and we believe it is time to revisit it.

The increment of the temperature of maximum density of water by addition of small amounts of tert-butanol: Experimental data and microscopic description revisited.

The temperature of maximum density, TMD, of aqueous solutions of tert-butanol has been experimentally determined in the pressure range of 0-300 bars and up to 0.025 tert-butanol mole fraction. At atmospheric pressure, this quantity increases for low alcohol mole fractions, reaches a maximum at intermediate concentrations, and then quickly falls. The new experimental results are basically in agreement with previous data in the literature by Wada and Umeda [G. Wada and S. Umeda, Bull. Chem. Soc. Jpn. 35, 646 (1962)], except at very low mole fractions, where these authors reported a stronger density anomaly. Our measurements also confirm the known effect of pressure, p, on the variation in the temperature of maximum density with respect to that of pure water, ΔTMD: this quantity increases with p over the whole composition range. In addition, molecular dynamics simulations were performed between 0 and 2000 bars and from 238 to 328 K using a recently proposed model for the tert-butanol/water system. It has been found that our model reproduces qualitatively the experimental behavior of the ΔTMD, but for pressures above 1000 bars. A detailed structural analysis showed that the addition of tert-butanol promotes the low density water structure, and this promotion is somewhat hampered as the temperature increases at high pressure (ΔTMD > 0) and mostly independent of temperature at low pressures (ΔTMD < 0). Our analysis shows that the ultimate factor determining changes in the TMD is the temperature dependence of the low density water structure enhancement. We have also carried out a local structure analysis in which in addition to solid-like structures, low density liquid water ones have also been considered.

Larval cryopreservation as new management tool for threatened clam fisheries.

Cryopreservation is the only reliable method for long-term storage of biological material that guarantees genetic stability. This technique can be extremely useful for the conservation of endangered species and restock natural populations for declining species. Many factors have negatively affected the populations of high economical value shellfish in Spain and, as a result, many are declining or threatened nowadays. This study was focused on early-life stages of Venerupis corrugata, Ruditapes decussatus and Ruditapes philippinarum to develop successful protocols to enhance the conservation effort and sustainable shellfishery resources. Firstly, common cryoprotecting agents (CPAs) were tested to select the suitable permeable CPA attending to toxicity. Cryopreservation success using different combinations of CPA solutions, increasing equilibrium times and larval stages was evaluated attending to survival and shell growth at 2 days post-thawing. Older clam development stages were more tolerant to CPA toxicity, being ethylene-glycol (EG) and Propylene-glycol (PG) the least toxic CPAs. CPA solution containing EG yielded the highest post-thawing survival rate and the increase of equilibration time was not beneficial for clam larvae. Cryopreservation of trochophores yielded around 50% survivorship, whereas over 80% of cryopreserved D-larvae were able to recover after thawing.

Spotting intruders: Species distribution models for managing invasive intertidal macroalgae.

Invasive macroalgae represent one of the major threats to marine biodiversity, ecosystem functioning and structure, as well as being important drivers of ecosystem services depletion. Many such species have become well established along the west coast of the Iberian Peninsula. However, the lack of information about the distribution of the invaders and the factors determining their occurrence make bioinvasions a difficult issue to manage. Such information is key to enabling the design and implementation of effective management plans. The present study aimed to map the current probability of presence of six invasive macroalgae: Grateloupia turuturu, Asparagopsis armata, Colpomenia peregrina, Sargassum muticum, Undaria pinnatifida, and Codium fragile ssp. fragile. For this purpose, an extensive field survey was carried out along the coast of the north-western Iberian Peninsula. Species distribution models (SDMs) were then used to map the presence probability of these invasive species throughout the study region on the basis of environmental and anthropogenic predictor variables. The southern Galician rias were identified as the main hotspots of macroalgal invasion, with a high probability of occurrence for most of the species considered. Conversely, the probability of presence on the Portuguese coast was generally low. Physico-chemical variables were the most important factors for predicting the distribution of invasive macroalgae contributing between 57.27 and 85.24% to the ensemble models. However, anthropogenic factors (including size of vessels, number of shipping lines, distance from ports, population density, etc.) considerably improved the estimates of the probability of occurrence for most of the target species. This study is one of the few to include anthropogenic factors in SDMs for invasive macroalgae. The findings suggest that management actions aimed at controlling these species should strengthen control and surveillance at ports, particularly in southern Galician rias. Early detection should be of main concern for risk assessment plans on the Portuguese coast.

Major challenges in cryopreservation of sea urchin eggs.

Cryopreservation of gametes, embryos and larvae of marine invertebrates has been investigated in many studies throughout the years. There are many favorable studies on sperm cryopreservation but oocytes are still under research as no successful results have been sustainably obtained for this type of cells. The preservation of both maternal and paternal gametes separately would provide a reliable source of genetic material for their application to conservation, aquaculture and fundamental research. Unfortunately to date, it has not been possible to cryopreserve eggs from marine organisms. The aim of this review is to go over the factors that have been historically considered as obstacles for oocyte cryopreservation in aquatic organisms and discern those that may specifically apply to eggs of the sea urchin Paracentrotus lividus.

Toxicity tests of cryoprotecting agents for Mytilus galloprovincialis (Lamark, 1819) early developmental stages.

Global aquaculture production of blue mussel has increased over last years. This work reaffirms the great potential of cryopreservation technique on mussel industry and overcome economic barriers a cause of a traditional and rudimentary management and continue growing. The aim of this work is to set some preliminary basis attending to toxicity of cryoprotecting agents (CPAs) on different development stages of Mytilus galloprovincialis as a start point to develop a stable cryopreservation protocol. Toxicity tests were carried out by using common CPAs (dimethyl-sulfoxide (Me2SO), glycerol, (GLY), propylene glycol (PG) and ethylene glycol (EG)) in a range from 0.5 to 3 M on fertilized egg, trochophore larva, and D-larva of Mytilus galloprovincialis. Results evidenced more resistance of older development stages to toxicity. Of all CPAs tested, toxicity testing highlights PG or EG as suitable CPAs for cryopreservation of early development stages; whereas D-larva was unaffected by any of the CPAs tested. Preliminary cryopreservation trials were developed to obtain information into cell cryoprotection. Further research should be focused on membrane permeability and other parameters, such as the balance between toxicity and cryoprotective effect of CPAs.

Alpha-2 macroglobulin in Alzheimer's disease: a marker of neuronal injury through the RCAN1 pathway.

Preclinical changes that precede the onset of symptoms and eventual diagnosis of Alzheimer's disease (AD) are a target for potential preventive interventions. A large body of evidence suggests that inflammation is closely associated with AD pathogenesis and may be a promising target pathway for such interventions. However, little is known about the association between systemic inflammation and preclinical AD pathophysiology. We first examined whether the acute-phase protein, alpha-2 macroglobulin (A2M), a major component of the innate immune system, was associated with cerebrospinal fluid (CSF) markers of neuronal injury in preclinical AD and risk of incident AD in the predictors of cognitive decline among normal individuals (BIOCARD) cohort. We find that A2M concentration in blood is significantly associated with CSF concentrations of the neuronal injury markers, tau and phosphorylated tau, and that higher baseline serum A2M concentration is associated with an almost threefold greater risk of progression to clinical symptoms of AD in men. These findings were replicated in the Alzheimer's Disease Neuroimaging (ADNI) study. Then, utilizing a systems level approach combining large multi-tissue gene expression datasets with mass spectrometry-based proteomic analyses of brain tissue, we identified an A2M gene network that includes regulator of calcineurin (RCAN1), an inhibitor of calcineurin, a well-characterized tau phosphatase. A2M gene and protein expression in the brain were significantly associated with gene and protein expression levels of calcineurin. Collectively these novel findings suggest that A2M is associated with preclinical AD, reflects early neuronal injury in the disease course and may be responsive to tau phosphorylation in the brain through the RCAN1-calcineurin pathway.

Midlife adiposity predicts earlier onset of Alzheimer's dementia, neuropathology and presymptomatic cerebral amyloid accumulation.

Understanding how midlife risk factors influence age at onset (AAO) of Alzheimer's disease (AD) may provide clues to delay disease expression. Although midlife adiposity predicts increased incidence of AD, it is unclear whether it affects AAO and severity of Alzheimer's neuropathology. Using a prospective population-based cohort, Baltimore Longitudinal Study of Aging (BLSA), this study aims to examine the relationships between midlife body mass index (BMI) and (1) AAO of AD (2) severity of Alzheimer's neuropathology and (3) fibrillar brain amyloid deposition during aging. We analyzed data on 1394 cognitively normal individuals at baseline (8643 visits; average follow-up interval 13.9 years), among whom 142 participants developed incident AD. In two subsamples of BLSA, 191 participants underwent autopsy and neuropathological assessment, and 75 non-demented individuals underwent brain amyloid imaging. Midlife adiposity was derived from BMI data at 50 years of age. We find that each unit increase in midlife BMI predicts earlier onset of AD by 6.7 months (P=0.013). Higher midlife BMI was associated with greater Braak neurofibrillary but not CERAD (Consortium to Establish a Registry for Alzheimer's Disease) neuritic plaque scores at autopsy overall. Associations between midlife BMI and brain amyloid burden approached statistical significance. Thus, higher midlife BMI was also associated with greater fibrillar amyloid measured by global mean cortical distribution volume ratio (P=0.075) and within the precuneus (left, P=0.061; right, P=0.079). In conclusion, midlife overweight predicts earlier onset of AD and greater burden of Alzheimer's neuropathology. A healthy BMI at midlife may delay the onset of AD.

Resultados de la reparación artroscópica mediante anclaje óseo sin nudo de lesiones del fibrocartílago triangular asociadas a fractura de extremidad distal de radio / Arthroscopic knotless anchor repair of triangular fibrocartilage in distal radius fracture

Introducción y objetivo. Las lesiones del fibrocartílago triangular (FCT) están asociadas en muchas ocasiones con inestabilidad de la articulación radiocubital distal. El tratamiento artroscópico de estas lesiones mejora el pronóstico funcional de los pacientes afectados. El objetivo del presente trabajo es evaluar los resultados funcionales y laborales de la reparación artroscópica con anclaje óseo sin nudo de lesiones del FCT asociadas a fracturas de extremidad distal del radio. Material y método. Estudio observacional, descriptivo entre noviembre de 2011 y enero de 2014 de 21 pacientes con fractura de radio distal asociada a lesiones tipo IB de Palmer (clases 2 y 3 de Atzei) del FCT tratados mediante reparación artroscópica con anclaje óseo sin nudo (PopLok® 2,8 mm, ConMed, EE. UU.). El tiempo medio de seguimiento fue de 18 meses. Analizamos los resultados funcionales (Mayo Wrist Score) y laborales. La edad media fue de 43,0 ± 8,8 años, con un 19% de mujeres. Se hallaron 5 casos con lesión escafolunar asociada. Resultados. La media obtenida en la escala funcional de Mayo Wrist Score fue de 83,4 ± 16,1 puntos. El tiempo de baja medio fue de 153,16 ± 48,5 días. La recuperación laboral sin limitaciones fue posible en el 89,5% de los casos. No se recogieron complicaciones postoperatorias. Conclusiones. La reparación artroscópica con anclaje óseo sin nudo de lesiones IB del FCT en pacientes con fractura de radio distal supone un método de tratamiento mínimamente invasivo, que mejora la tensión en la reparación y evita el posterior destensado, en nuestra experiencia, con baja tasa de complicaciones y buenos resultados funcionales y laborales (AU)

Introduction and objective. Lesions of triangular fibrocartilage (TFC) are associated with distal radioulnar joint instability. Arthroscopic treatment of these lesions improves functional outcome of affected patients. The aim of the present work is to evaluate functional and occupational outcome of TCF repair using an arthroscopic knotless anchor device in patients with associated distal radius fracture. Material and methods. An observational, descriptive study was carried out between November 2011 and January 2014 including 21 patients with distal radius fracture and Palmer 1 B lesions of TCF (Atzei class 2 and 3) that were treated by arthroscopic knotless anchor (PopLok® 2,8 mm, ConMed, USA). Mean follow-up was 18 months. Functional (Mayo Wrist Score) and occupational outcome results were analyzed. Mean age of the group was 43.0 ± 8.8 years, with 19% of the patients being female. There was an associated scapholunate lesion in 5 cases. Results. Functional results reached a mean of 83.4 ± 16.1 points onMayo Wrist Score. Mean sick-leave time was 153.16 ± 48.5 days. Complete occupational reintegration was reached in 89.5% of cases. There were no postoperative complications. Conclusions. Arthroscopic knotless anchor repair of 1 B TFC tears is a minimally invasive method of treatment that improves tension of fixation, avoiding subsequent loosen, in our experience, with few complications and good functional and occupational results (AU)

Effectiveness of egg yolk immunoglobulin against the intracellular salmonid pathogen Piscirickettsia salmonis.

AIMS: To produce and characterize egg yolk immunoglobulin (IgY) against the fish intracellular pathogen Piscirickettsia salmonis as well as to evaluate the antibacterial activity of IgY in vitro and the availability in the serum of fish immunized orally. METHODS AND RESULTS: Specific IgY was produced by immunizing hens with P. salmonis proteins. The IgY was obtained from egg yolks using the ammonium sulphate precipitation method and it was characterized by SDS-PAGE, Western-blot and ELISA, demonstrating that anti-P. salmonis IgY strongly reacted specifically against P. salmonis proteins. In an in vitro neutralization assay, IgY inhibited the growth of P. salmonis in liquid medium at concentrations ranging from 128 to 256 µg ml(-1) in a dose-dependent manner. Interestingly, IgY against P. salmonis also generates a strong protective effect on the infection of P. salmonis in salmon head kidney-1 cells. In addition, the bacteriostatic function of IgY appears to result possibly from agglutination by the interaction of IgY with surface components of the pathogen. Finally, to confirm this IgY as an alternative for salmonid treatment, Atlantic salmon (Salmo salar) specimens were orally inoculated with IgY. The analysis of the sera demonstrates that IgY was effectively transported by fish intestine and that this immunoglobulins maintains its properties and recognizes several proteins of P. salmonis up to 12 h after inoculation of IgY against P. salmonis. CONCLUSIONS: Specific IgY effectively inhibited the growth of P. salmonis and this immunoglobulin can be released in the Atlantic salmon sera when administered orally to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose that this specific IgY against this fastidious micro-organism could be a useful strategy for the treatment of piscirickettsiosis.

Arthroscopic knotless anchor repair of triangular fibrocartilage in distal radius fracture.

INTRODUCTION AND OBJECTIVE: Lesions of triangular fibrocartilage (TFC) are associated with distal radioulnar joint instability. Arthroscopic treatment of these lesions improves functional outcome of affected patients. The aim of the present work is to evaluate functional and occupational outcome of TCF repair using an arthroscopic knotless anchor device in patients with associated distal radius fracture. MATERIAL AND METHODS: An observational, descriptive study was carried out between November 2011 and January 2014 including 21 patients with distal radius fracture and Palmer 1B lesions of TCF (Atzei class 2 and 3) that were treated by arthroscopic knotless anchor (PopLok® 2,8mm, ConMed, USA). Mean follow-up was 18 months. Functional (Mayo Wrist Score) and occupational outcome results were analyzed. Mean age of the group was 43.0±8.8 years, with 19% of the patients being female. There was an associated scapholunate lesion in 5 cases. RESULTS: Functional results reached a mean of 83.4±16.1 points onMayo Wrist Score. Mean sick-leave time was 153.16±48.5 days. Complete occupational reintegration was reached in 89.5% of cases. There were no postoperative complications. CONCLUSIONS: Arthroscopic knotless anchor repair of 1B TFC tears is a minimally invasive method of treatment that improves tension of fixation, avoiding subsequent loosen, in our experience, with few complications and good functional and occupational results.

Short- and long-term outcomes of every graft recovered during a multi-organ procurement procedure including the intestine.

BACKGROUND: The development of intestinal transplant (Tx) programs introduces thymoglobulin donor treatment as well as an almost complete warm dissection of the abdominal organs to allocate them to different recipients. Our aim is to assess the reproducibility and feasibility of the surgical technique of multi-organ procurement with the use of thymoglobulin donor pre-treatment and report the short- and long-term outcomes of every graft harvested as part of multi-organ procurement (MTOp), including the intestine. METHODS: Data were collected of all organs harvested from MTOp, including the intestines allocated to our center from March 2006 to July 2011. Data from 92 recipients and 116 organs procured from 29 MTOp were analyzed. Twelve hearts, 2 lungs, and 1 cardio-pulmonary block were transplanted; primary graft dysfunction developed in 4 of the 12 hearts and in the cardio-pulmonary block. RESULTS: The survival rate was 75% and 100% for hearts and lungs, respectively. Nineteen livers, 9 kidney-pancreas, 19 kidneys, and 29 intestines were transplanted. Delayed graft function (DGF) of the pancreas developed in 3 of 9 kidney-pancreas, and the other 3 exhibited DGF of the kidney; 4 of 19 Tx kidneys had DGF. The survival was 84%, 78%, 95%, and 65.5% for livers, kidney-pancreas, kidneys, and intestines, respectively. CONCLUSIONS: Organs procured during MTOp including the intestine can be safely used, increasing organ availability and transplant applicability without compromising allocation, quality, and long-term results of the non-intestinal-procured organs.

Recurrent cerebral venous thrombosis associated with heterozygote methylenetetrahydrofolate reductase C677T mutation and sickle cell trait without homocysteinemia: an autopsy case report and review of literature.

Elevated blood homocysteine concentration and certain genetic mutations have been associated with increased risk for developing arterial and venous thrombosis. A common mutation of methylenetetrahydrofolate reductase, MTHFR C677T, has been associated with elevated homocysteine concentration and increased risk for developing thrombosis in homozygote carriers. Heterozygote carriers for this gene mutation, if associated with other major or minor risk factors for thrombophilia, appear to be prone to develop thrombosis. A postmortem genetic testing for common mutations resulting in thrombophilia should be performed in all individuals who die as a result of thrombosis, regardless of predisposing risk factors, to determine the true prevalence of mutations in these individuals, and to assess the true role of a certain mutation, such as heterozygote MTHFR C677T, in the pathogenesis of thrombosis. Postmortem genetic testing for common mutations associated with thrombophilia in selected cases has potentially life-saving importance to surviving family members. We report a case of recurrent cerebral venous thrombosis in a 19 year old male with history of sickle cell trait, obesity, and high normal blood homocysteine, who was heterozygote for MTHFR C677T mutation.