Association of Childhood Exposure to School Racial Segregation with Late-Life Cognitive Outcomes among Older Americans.

IMPORTANCE: Disparities in cognition, including dementia occurrence, persist between White and Black older adults, and are possibly influenced by early educational differences stemming from structural racism. However, the relationship between school racial segregation and later-life cognition remains underexplored. OBJECTIVE: To investigate the association between childhood contextual exposure to school racial segregation and cognitive outcomes in later life. DESIGN SETTING AND PARTICIPANTS: Data from 16,625 non-Hispanic White (hereafter, White) and 3,335 non-Hispanic Black (hereafter, Black) Americans aged 65 or older were analyzed from the Health and Retirement Study. EXPOSURES: State-level White-Black dissimilarity index for public elementary schools in the late 1960s (range: 0-100) was used to measure school segregation. States were categorized into high segregation (383.6) and low segregation (<83.6) based on the top quintile. MAIN OUTCOMES AND MEASURES: Cognitive scores, cognitive impairment (with or without dementia), and dementia were assessed using the Telephone Interview for Cognitive Status (TICS) and proxy assessment. Multilevel regression analyses were conducted, adjusting for demographic covariates, socioeconomic status, and health factors. Stratified analyses by race were performed. RESULTS: The mean (SD) age of participants was 78.5 (5.7) years, and 11,208 (56.2%) were female. Participants exposed to high segregation exhibited lower cognitive scores (12.6 vs. 13.6; P<0.001) and higher prevalence of cognitive impairment (50.8% vs 41.4%; P<0.001) and dementia (26.0% vs. 19.5%; P<0.001), compared to those with low segregation exposure. Multilevel analyses revealed a significant negative association between school segregation and later-life cognitive even after adjusting sequentially for potential confounders, and these associations were stronger among Black than White participants. Notably, in the fully adjusted model, Black participants exposed to high segregation displayed significantly lower cognitive scores (-0.51; 95% CI: -0.94, -0.09) and higher likelihood of cognitive impairment (adjusted Odds Ratio [aOR]: 1.45, 95% CI: 1.22, 1.72) and dementia (aOR: 1.31, 95% CI: 1.06, 1.63). CONCLUSIONS AND RELEVANCE: Our study underscores that childhood exposure to state-level school segregation is associated with late-life cognition, especially for Black Americans. Given the rising trend of school segregation in the US, educational policies aimed at reducing segregation are crucial to address health inequities. Clinicians can leverage patients' early-life educational circumstances to promote screening, prevention, and management of cognitive disorders.

Association of Family Support With Lower Modifiable Risk Factors for Dementia Among Cognitively Impaired Older Adults.

OBJECTIVES: Cognitive impairment poses considerable challenges among older adults, with the role of family support becoming increasingly crucial. This study examines the association of children's residential proximity and spousal presence with key modifiable risk factors for dementia in cognitively impaired older adults. METHODS: We analyzed 14,600 individuals (35,165 observations) aged 50 and older with cognitive impairment from the Health and Retirement Study (1995-2018). Family support was categorized by spousal presence and children's residential proximity. Modifiable risk factors, including smoking, depressive symptoms, and social isolation, were assessed. Associations between family support and the modifiable risk factors were determined using mixed-effects logistic regressions. RESULTS: A significant proportion of older adults with cognitive impairment lacked access to family support, with either no spouse (46.9%) or all children living over 10 miles away (25.3%). Those with less available family support, characterized by distant-residing children and the absence of a spouse, had a significantly higher percentage of smoking, depressive symptoms, and social isolation. Moreover, we revealed a consistent gradient in the percentage of the risk factors by the degree of family support. Relative to older adults with a spouse and co-resident children, those without a spouse and with all children residing further than 10 miles displayed the highest percentage of the risk factors. These findings were robust to various sensitivity analyses. CONCLUSIONS: Family support from spouses and nearby children serves as a protective factor against modifiable dementia risk factors in cognitively impaired older adults. Policies that strengthen family and social support may benefit this population.

Early-Life Circumstances and Racial Disparities in Cognition Among Older Adults in the US.

Importance: Given the critical role of neurocognitive development in early life, understanding the association between early-life circumstances and racial disparities in cognition has important implications. Objective: To assess whether racial differences in early-life circumstances are collectively and individually associated with racial disparities in late-life cognition among older adults in the US. Design, Setting, and Participants: This cross-sectional study used comprehensive life history data from the Health and Retirement Study, a nationally representative survey of US adults 50 years or older. Data analyses were performed from August 9, 2022, to January 20, 2024. Main Outcomes and Measures: Racial differences in early-life circumstances and racial disparities in late-life cognition were investigated using a Blinder-Oaxaca decomposition regression model. Cognitive outcomes, including cognitive score and cognitive impairment, were evaluated using the Telephone Interview for Cognitive Status. Early-life educational experiences were primary explanatory variables; early-life cohort, regional, financial, health, trauma, family relationship factors, and educational attainment were additional explanatory variables; demographic and genetic factors were covariates. Results: The study sample comprised 9015 participants; 1634 non-Hispanic Black (hereafter, Black) individuals (18.1%) and 7381 non-Hispanic White (hereafter, White) individuals (81.9%). Among Black participants, the mean (SD) age was 69.2 (9.2) years and 1094 (67.0%) were women. Among White participants, the mean (SD) age was 73.2 (10.1) years and 4410 (59.7%) were women. Cognitive scores (scale, 0-27) were significantly lower among Black participants (13.5 [95% CI, 13.3-13.7] points) than among White participants (15.8 [95% CI, 15.7-15.9] points), while the prevalence of cognitive impairment (cognitive score <12) was significantly higher among Black participants (33.6 [95% CI, 31.3-35.9] percentage points [ppt]) than among White participants (16.4 [95% CI, 15.6-17.2] ppt). Substantial racial differences were observed in early-life circumstances. Overall, differences in early-life circumstances were associated with 61.5% of the racial disparities in cognitive score (1.4 [95% CI, 0.88-2.0] points), and 82.3% of the racial disparities in cognitive impairment (14.2 [95% CI, 8.8-19.5] ppt), respectively. In multivariable analyses, early-life educational experiences were associated with 35.2% of the disparities in cognitive score and 48.6% in cognitive impairment. Notably, school racial segregation (all segregated schooling before college) was associated with 28.8% to 39.7% of the racial disparities in cognition. These findings were consistent in a series of sensitivity analyses. Conclusions and Relevance: The findings of this cross-sectional study suggest that less favorable early-life circumstances are associated with clinically meaningful racial disparities in late-life cognition. Policies that improve educational equity have the potential to reduce racial disparities in cognition in older ages. Clinicians may leverage early-life circumstances to promote the screening, prevention, and interventions of cognitive impairment more efficiently, thereby promoting health equity.

The Long-Term Impacts of Dementia on Preventive Care Utilization and Health Behaviors.

Dementia has large impact on individuals' decision making, independent living, and wellbeing. Identifying early signals of dementia risk may offer people more time to prepare for the future, helping to delay the onset or slow the progression of dementia. Using the 1995-2018 waves of Health and Retirement Study, we offer novel evidence on the impacts of dementia on a rich set of preventive care utilization and health behaviors. Leveraging both within- and between-individual variations in an event study design, we characterize long-term dynamic changes in preventive care and health behaviors relative to the incidence of dementia and find early behavioral indicators of the disorder. We show that relative to the group of people who never develop dementia during the study periods, people with dementia have consistent and escalating declines in the use of cholesterol test, dental visit, prostate test and mammogram around the incidence of dementia. Significant declines are also found in physical activities and social engagement. Importantly, we demonstrate that the behavioral changes can occur up to 6 years before the incidence of dementia; and these patterns are absent in other chronic or acute conditions. The results are robust to sample selection, model specification, and the further control of aging and cohort effects. Overall, our findings highlight the salient impact of dementia risk on preventive care utilization and health behaviors, which may increase individuals' vulnerability to health shocks. Detecting early signals of dementia and facilitating targeted interventions are thus called for to prevent individuals from adverse behavioral and health consequences.

Children's Residential Proximity, Spousal Presence and Modifiable Risk Factors for Dementia among Older Adults with Cognitive Impairment.

Background: Cognitive impairment in older adults poses considerable challenges, and the role of family support becomes increasingly crucial. This study aims to examine the impact of children's residential proximity and spousal presence on the key modifiable risk factors for dementia among older adults with cognitive impairment. Methods: Utilizing the Health and Retirement Study (HRS) data from 1995 to 2018, we analyzed 14,731 participants (35,840 person-waves) aged 50 and older with cognitive impairment. Family support was characterized based on the presence of a spouse and residential proximity to children. Smoking, depressive symptoms and social isolation were included as the key modifiable risk factors for dementia identified in later life. Using mixed-effects logistic regressions, associations between access to family support and the modifiable risk factors were determined, adjusting for various socio-demographic and health-related factors. Results: Significant associations were found between access to family support and modifiable risk factors for dementia. Cognitively impaired older adults with less available family support, characterized by distant-residing children and the absence of a spouse, had significantly higher risks of smoking, depressive symptoms, and social isolation. Moreover, we revealed a consistent gradient in the prevalence of the risk factors based on the degree of family support. Relative to older adults with a spouse and co-resident children, those without a spouse and with all children residing further than 10 miles displayed the highest risks of smoking, depressive symptoms, and social isolation. Conclusion: Access to family support, particularly from spouses and proximate children, plays a protective role against key modifiable risk factors for dementia in older adults with cognitive impairment. The findings highlight the need for bolstering family and social support systems to enhance the well-being of this vulnerable population.

Place of Birth and Cognition among Older Americans: Findings from the Harmonized Cognitive Assessment Protocol.

Objectives: Growing evidence suggests that place of birth (PoB) and related circumstances may have long-lasting and multiplicative contributions to various later-life outcomes. However, the specific contributions to different domains of cognitive function in late life remain less understood. This study aimed to investigate the extent to which PoB contribute to a wide range of later-life cognitive outcomes. Methods: A nationally representative sample of Americans aged 65 and older (N=3,216) from the Health and Retirement Study (HRS) Harmonized Cognitive Assessment Protocol (HCAP) was utilized. Cognitive outcomes were assessed in HCAP and linked to HRS state-level PoB data to explore the contribution of birthplace to later-life cognitive disparities. Regression-based Shapley decompositions were employed to quantify this contribution. Results: PoB significantly contributed to all assessed cognitive outcomes including memory, executive function, language and fluency, visuospatial function, orientation, global cognitive performance, cognitive impairment and dementia. Geographic disparities in cognitive outcomes were evident, with individuals born in US southern states and foreign-born individuals performing worse than those born in other states. PoB overall accounted for 2.4-13.9% of the total variance in cognition after adjusting for age and sex. This contribution reduced by half when adjusting for a rich set of sociodemographic and health factors over the life course, but PoB still independently explained 2.0-7.1% of the total variance in cognition. Discussion: PoB has lasting contributions to later-life cognitive health, with significant geographic disparities observed. Addressing these disparities requires promoting more equalized place-based policies, resources, and early-life environments to improve health equities over the life course.

Self-perceived memory is negatively associated with chronic disease awareness: Evidence from blood biomarker data.

Background and objectives: Cognitive misperception contributed to poor decision-making; yet their impact on health-related decisions is less known. We examined how self-perceived memory was associated with chronic disease awareness among older Chinese adults. Research design and methods: Data were obtained from the China Health and Retirement Longitudinal Study. Nationally representative blood biomarkers were collected in 2015 to identify participants' dyslipidemia and diabetes status. Among participants with biomarker identified dyslipidemia or diabetes, disease awareness was defined as self-reported diagnosis of the conditions as of 2018. The association of self-perceived memory with chronic disease awareness was determined by weighted multivariate logistic regressions adjusting for cognitive ability and covariates. Results: Among 4578 adults aged 60 and over, 1442 and 759 individuals were identified having dyslipidemia and diabetes, with proportions of disease awareness being 38.0% and 58.1%, respectively. The proportions were lower for individuals with better self-perceived memory and those with more impaired cognitive ability, showing opposite patterns. Adjusting for cognitive ability and covariates, self-perceived memory was negatively associated with the dyslipidemia (OR = 0.80, 95%CI: 0.63-1.02) and diabetes (OR = 0.71, 95%CI: 0.55-0.92) awareness. In particular, older adults with the highest level of self-perceived memory had significantly lower disease awareness as compared to those with the lowest level of self-perceived memory (OR = 0.51, 95%CI: 0.28-0.94 for dyslipidemia; and OR = 0.42, 95%CI: 0.21-0.84 for diabetes). The negative association was robust to adjusting for alternative cognitive measures, and was stronger for individuals with rural status, lower education, or living without children. Discussion and implications: Cognitive misperception poses great challenges to chronic disease awareness. Targeted interventions and supports are needed, particularly for those more disadvantaged.

Geographic Variation in Inpatient Care Utilization, Outcomes and Costs for Dementia Patients Aged 65 Years or Older - China, 2017-2019.

What is already known about this topic?: Dementia leads public health issues worldwide. China has the largest population of adults living with dementia in the world, imposing increasing burdens on the public health and healthcare systems. Despite improved access to health services, inadequate and uneven dementia management remains common. What is added by this report?: The report documents the provincial-level geographic patterns in healthcare utilization, outcomes, and costs for patients hospitalized for dementia in China. Regional patterns demonstrate gaps in equity and efficiency of dementia care and management for dementia patients. What are the implications for public health practice?: Public health policy and practices should consider geographic disparities in disease burden and healthcare provision to promote equitable allocation of resources for dementia care throughout China.

Adverse childhood circumstances and cognitive function in middle-aged and older Chinese adults: Lower level or faster decline?

We examine the long-term relationship between childhood circumstances and cognitive aging. In particular, we differentiate the level of cognitive deficit from the rate of cognitive decline. Applying a linear mixed-effect model to three waves of China Health and Retirement Longitudinal Surveys (CHARLS 2011, 2013, 2015) and matching cognitive outcomes to CHARLS Life History Survey (2014), we find that key domains of childhood circumstances, including family socioeconomic status (SES), neighborhood cohesion, friendship, and health conditions, are significantly associated with both the level of cognitive deficit and the rate of decline. In contrast, childhood neighborhood safety only affects the level of cognitive deficit. Childhood relationship with mother only affects the rate of cognitive decline. The effects of adverse childhood circumstances are generally larger on level of cognitive deficit than on rate of cognitive decline. Moreover, education plays a more important role in mediating the relationships compared to other later-life factors. These findings suggest that exposure to disadvantaged childhood circumstances can exacerbate cognitive deficit as well as cognitive decline over time, which may be partially ameliorated by educational attainment.

Regulators of complement activity mediate inhibitory mechanisms through a common C3b-binding mode.

Regulators of complement activation (RCA) inhibit complement-induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i-iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b-binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease-related mutations and immune evasion.

Complement C3dg-mediated erythrophagocytosis: implications for paroxysmal nocturnal hemoglobinuria.

The clinical management of paroxysmal nocturnal hemoglobinuria (PNH), a rare but life-threatening hematologic disease, has fundamentally improved with the introduction of a therapeutic that prevents complement-mediated intravascular hemolysis. However, a considerable fraction of PNH patients show insufficient treatment response and remain transfusion dependent. Because the current treatment only prevents C5-induced lysis but not upstream C3 activation, it has been speculated that ongoing opsonization with C3 fragments leads to recognition and phagocytosis of PNH erythrocytes by immune cells. Here, for the first time, we provide experimental evidence for such extravascular hemolysis and demonstrate that PNH erythrocytes from anti-C5-treated patients are phagocytosed by activated monocytes in vitro. Importantly, we show that this uptake can be mediated by the end-stage opsonin C3dg, which is not traditionally considered a phagocytic marker, via interaction with complement receptor 3 (CR3). Interaction studies confirmed that C3dg itself can act as a ligand for the binding domain of CR3. The degree of C3dg-mediated erythrophagocytosis in samples from different PNH patients correlated well with the individual level of C3dg opsonization. This finding may guide future treatment options for PNH but also has potential implications for the description and management of other complement-mediated diseases.

Peptide inhibitors of C3 activation as a novel strategy of complement inhibition for the treatment of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis due to the lack of CD55 and CD59 on affected erythrocytes. The anti-C5 antibody eculizumab has proven clinically effective, but uncontrolled C3 activation due to CD55 absence may result in opsonization of erythrocytes, possibly leading to clinically meaningful extravascular hemolysis. We investigated the effect of the peptidic C3 inhibitor, compstatin Cp40, and its long-acting form (polyethylene glycol [PEG]-Cp40) on hemolysis and opsonization of PNH erythrocytes in an established in vitro system. Both compounds demonstrated dose-dependent inhibition of hemolysis with IC50 ∼4 µM and full inhibition at 6 µM. Protective levels of either Cp40 or PEG-Cp40 also efficiently prevented deposition of C3 fragments on PNH erythrocytes. We further explored the potential of both inhibitors for systemic administration and performed pharmacokinetic evaluation in nonhuman primates. A single intravenous injection of PEG-Cp40 resulted in a prolonged elimination half-life of >5 days but may potentially affect the plasma levels of C3. Despite faster elimination kinetics, saturating inhibitor concentration could be reached with unmodified Cp40 through repetitive subcutaneous administration. In conclusion, peptide inhibitors of C3 activation effectively prevent hemolysis and C3 opsonization of PNH erythrocytes, and are excellent, and potentially cost-effective, candidates for further clinical investigation.

Rational engineering of a minimized immune inhibitor with unique triple-targeting properties.

Inadequate control of the complement system is the underlying or aggravating factor in many human diseases. Whereas treatment options that specifically target the alternative pathway (AP) of complement activation are considered highly desirable, no such option is available in the clinic. In this study, we present a successful example of protein engineering, guided by structural insight on the complement regulator factor H (FH), yielding a novel complement-targeted therapeutic (mini-FH) with clinical potential. Despite a 70% reduction in size, mini-FH retained and in some respects exceeded the regulatory activity and cell surface-recognition properties of its parent protein FH, including the recently described recognition of sites of oxidative stress. Importantly, the chosen design extended the functional spectrum of the inhibitor, as mini-FH showed increased binding to the surface-bound opsonins iC3b and C3dg when compared with FH. Thus, mini-FH is equipped with a unique and clinically valuable triple-targeting profile toward diseased host cells, through its binding to sites of ongoing complement activation, markers of oxidative damage, and host surface-specific polyanions. When assessed in a clinically relevant AP-mediated disease model of paroxysmal nocturnal hemoglobinuria, mini-FH largely outperformed FH and indicated advantages over clinically evaluated AP inhibitors. Thus, the rational engineering of a streamlined FH construct not only provided insight into the function of a key complement regulator, but also yielded a novel inhibitor that combines a triple-targeting approach with high AP-specific inhibitory activity (IC50 ~ 40 nM), which may pave the way toward new options for the treatment of complement-mediated diseases.

A structurally dynamic N-terminal helix is a key functional determinant in staphylococcal complement inhibitor (SCIN) proteins.

Complement is a network of interacting circulatory and cell surface proteins that recognizes, marks, and facilitates clearance of microbial invaders. To evade complement attack, the pathogenic organism Staphylococcus aureus expresses a number of secreted proteins that interfere with activation and regulation of the complement cascade. Staphylococcal complement inhibitors (SCINs) are one important class of these immunomodulators and consist of three active members (SCIN-A/-B/-C). SCINs inhibit a critical enzymatic complex, the alternative pathway C3 convertase, by targeting a functional "hot spot" on the central opsonin of complement, C3b. Although N-terminal truncation mutants of SCINs retain complement inhibitory properties, they are significantly weaker binders of C3b. To provide a structural basis for this observation, we undertook a series of crystallographic and NMR dynamics studies on full-length SCINs. This work reveals that N-terminal SCIN domains are characterized by a conformationally dynamic helical motif. C3b binding and functional experiments further demonstrate that this sequence-divergent N-terminal region of SCINs is both functionally important and context-dependent. Finally, surface plasmon resonance data provide evidence for the formation of inhibitor·enzyme·substrate complexes ((SCIN·C3bBb)·C3). Similar to the (SCIN·C3bBb)(2) pseudodimeric complexes, ((SCIN·C3bBb)·C3) interferes with the interaction of complement receptors and C3b. This activity provides an additional mechanism by which SCIN couples convertase inhibition to direct blocking of phagocytosis. Together, these data suggest that tethering multi-host protein complexes by small modular bacterial inhibitors may be a global strategy of immune evasion used by S. aureus. The work presented here provides detailed structure-activity relationships and improves our understanding of how S. aureus circumvents human innate immunity.

Diversity in the C3b [corrected] contact residues and tertiary structures of the staphylococcal complement inhibitor (SCIN) protein family.

To survive in immune-competent hosts, the pathogen Staphylococcus aureus expresses and secretes a sophisticated array of proteins that inhibit the complement system. Among these are the staphylococcal complement inhibitors (SCIN), which are composed of three active proteins (SCIN-A, -B, and -C) and one purportedly inactive member (SCIN-D or ORF-D). Because previous work has focused almost exclusively on SCIN-A, we sought to provide initial structure/function information on additional SCIN proteins. To this end we determined crystal structures of an active, N-terminal truncation mutant of SCIN-B (denoted SCIN-B18-85) both free and bound to the C3c fragment of complement component C3 at 1.5 and 3.4 Å resolution, respectively. Comparison of the C3c/SCIN-B18-85 structure with that of C3c/SCIN-A revealed that both proteins target the same functional hotspot on the C3b/C3c surface yet harbor diversity in both the type of residues and interactions formed at their C3b/C3c interfaces. Most importantly, these structures allowed identification of Arg44 and Tyr51 as residues key for SCIN-B binding to C3b and subsequent inhibition of the AP C3 convertase. In addition, we also solved several crystal structures of SCIN-D to 1.3 Å limiting resolution. This revealed an unexpected structural deviation in the N-terminal α helix relative to SCIN-A and SCIN-B. Comparative analysis of both electrostatic potentials and surface complementarity suggest a physical explanation for the inability of SCIN-D to bind C3b/C3c. Together, these studies provide a more thorough understanding of immune evasion by S. aureus and enhance potential use of SCIN proteins as templates for design of complement targeted therapeutics.

The natural resistance-associated macrophage protein from the protozoan parasite Perkinsus marinus mediates iron uptake.

Microbial pathogens succeed in acquiring essential metals such as iron and manganese despite their limited availability because of the host's immune response. The eukaryotic natural resistance-associated macrophage proteins mediate uptake of divalent metals and, during infection, may compete directly for metal acquisition with the pathogens' transporters. In this study, we characterize the Nramp gene family of Perkinsus marinus, an intracellular parasite of the eastern oyster, and through yeast complementation, we demonstrate for the first time for a protozoan parasite that Nramp imports environmental Fe. Three PmNramp isogenes differ in their exon-intron structures and encode transcripts that display a trans splicing leader at the 5' end. The protein sequences share conserved properties predicted for the Nramp/Solute carrier 11 (Slc11) family, such as 12-transmembrane segment (TMS) topology (N- and C-termini cytoplasmic) and preferential conservation of four TMS predicted to form a pseudosymmetric proton/metal symport pathway. Yeast fet3fet4 mutant complementation assays showed iron transport activity for PmNramp1 and a fusion chimera of the PmNramp3 hydrophobic core and PmNramp1 N- and C-termini. PmNramp1 site-directed mutagenesis demonstrated that Slc11 invariant and predicted pseudosymmetric motifs (TMS1 Asp-Pro-Gly and TMS6 Met-Pro-His) are key for transport function. PmNramp1 TMS1 mutants D76E, G78A, and D76E/G78A prevented membrane protein expression, while TMS6 M250A, H252Y, and M250A/H252Y specifically abrogated Fe uptake; the TMS6 H252Y mutation also correlates with divergence from Nramp specificity for divalent metals.

Transfection of the protozoan parasite Perkinsus marinus.

Ongoing efforts for sequencing the genome of the protozoan parasite Perkinsus marinus, together with functional genomic initiatives, have continued to provide invaluable information about genes and metabolic pathways that not only will increase our understanding of its biology, but also have the potential to reveal useful targets for intervention. The lack of molecular tools for the functional characterization of genes of interest, however, has hindered progress in this regard. Here we report the development and validation of transfection methodology for this parasite. We first selected from our P. marinus EST collection a highly expressed gene, which we designated "MOE" (PmMOE), to which we fused at the C-terminus the enhanced green fluorescent protein (GFP) as a reporter gene (pPmMOE-GFP). The exogenous DNA was introduced into the trophozoite stage of the parasite by electroporation using the Nucleofector technology. The transfection efficiency was 37.8% with fluorescence detected as early as 14 h after electroporation, with the transfectants still remaining fluorescent after 8 months even in the absence of drug selection. The 5' flanking region was essential for transcription; constructs with 100 and 204 bp flanking the transcription start site also drove transcription effectively. Polymerase chain reaction (PCR) and Southern blot analyses was consistent with integration by non-homologous recombination. This transfection technique, the first one reported for a member of the Perkinsozoa, provides a new tool for studies of gene regulation and expression, protein targeting, and protein-protein interactions, and should significantly contribute to gain further insight into the biology of Perkinsus spp.