Older Adult Compendium of Physical Activities: Energy costs of human activities in adults aged 60 and older.

PURPOSE: To describe the development of a Compendium for estimating the energy costs of activities in adults ≥60 years (OA Compendium). METHODS: Physical activities (PAs) and their metabolic equivalent of task (MET) values were obtained from a systematic search of studies published in 4 sport and exercise databases (PubMed, Embase, SPORTDiscus (EBSCOhost), and Scopus) and a review of articles included in the 2011 Adult Compendium that measured PA in older adults. MET values were computed as the oxygen cost (VO2, mL/kg/min) during PA divided by 2.7 mL/kg/min (MET60+) to account for the lower resting metabolic rate in older adults. RESULTS: We identified 68 articles and extracted energy expenditure data on 427 PAs. From these, we derived 99 unique Specific Activity codes with corresponding MET60+ values for older adults. We developed a website to present the OA Compendium MET60+ values: https://pacompendium.com. CONCLUSION: The OA Compendium uses data collected from adults ≥60 years for more accurate estimation of the energy cost of PAs in older adults. It is an accessible resource that will allow researchers, educators, and practitioners to find MET60+ values for older adults for use in PA research and practice.

2024 Adult Compendium of Physical Activities: A third update of the energy costs of human activities.

BACKGROUND: The Compendium of Physical Activities was published in 1993 to improve the comparability of energy expenditure values assigned to self-reported physical activity (PA) across studies. The original version was updated in 2000, and again in 2011, and has been widely used to support PA research, practice, and public health guidelines. METHODS: This 2024 update was tailored for adults 19-59 years of age by removing data from those ≥60 years. Using a systematic review and supplementary searches, we identified new activities and their associated measured metabolic equivalent (MET) values (using indirect calorimetry) published since 2011. We replaced estimated METs with measured values when possible. RESULTS: We screened 32,173 abstracts and 1507 full-text papers and extracted 2356 PA energy expenditure values from 701 papers. We added 303 new PAs and adjusted 176 existing MET values and descriptions to reflect the addition of new data and removal of METs for older adults. We added a Major Heading (Video Games). The 2024 Adult Compendium includes 1114 PAs (912 with measured and 202 with estimated values) across 22 Major Headings. CONCLUSION: This comprehensive update and refinement led to the creation of The 2024 Adult Compendium, which has utility across research, public health, education, and healthcare domains, as well as in the development of consumer health technologies. The new website with the complete lists of PAs and supporting resources is available at https://pacompendium.com.

Parent factors associated with BMI, diet, and physical activity of adolescents with intellectual and developmental disabilities.

BACKGROUND: Adolescents with intellectual and developmental disabilities (IDD) experience overweight and obesity (OW/OB) up to 1.8 times the rate of their typically developing peers. Parents may influence adolescent weight management behaviors in this population, but the association between parent factors and adolescent weight management behaviors is unclear. OBJECTIVE: To examine the associations between parent BMI and sociodemographic characteristics with adolescents' BMI, diet quality, daily energy intake, moderate to vigorous physical activity (MVPA), and sedentary behavior. METHODS: This study analyzed baseline data from an 18-month randomized controlled weight loss trial for adolescents with IDD. We assessed parent BMI (kg/m2) and sociodemographic factors, and adolescent BMI z-score, MVPA, sedentary time, daily energy intake, and diet quality. Associations between parent and adolescent factors were assessed with Pearson, Spearman or Kendall Tau-b correlations; mean differences for categorical outcomes were assessed with independent samples t-tests/Mann-Whitney U tests or ANOVA/Kruskall-Wallis tests. RESULTS: Ninety-five adolescent and parent dyads were included. Parent BMI was positively correlated with adolescent BMI z-score (n = 94: rs = 0.37, p < 0.01). Household income was inversely correlated with adolescent BMI z-score (n = 95: Tb = -0.18, p = 0.02). Parents with less than a bachelor's degree had adolescents with higher BMI z-scores than those with bachelor's or higher (2.1 ± 0.5 vs. 1.8 ± 0.5, p = 0.02) as well as higher sedentary behavior (n = 28, 515.2 ± 102.6 min/day vs. n = 40, 463.9 ± 148.1 min/day, p = 0.02). CONCLUSION: We found parent BMI, income, and education associated with adolescent BMI z-score. These findings contribute to the sparse literature on parental factors associated with OW/OB in this population. CLINICAL TRIALS NUMBER: NCT02561754.

The prevalence of obesity and lifestyle behaviors of parents of youth with intellectual and developmental disabilities.

BACKGROUND: Parents of youth with intellectual and developmental disabilities (IDD) may have a higher prevalence of overweight and obesity and poorer weight management behaviors compared to the general population. OBJECTIVE: To describe the prevalence of overweight/obesity and related socioeconomic and lifestyle factors including diet quality, physical activity, and reported health habits in parents of youth with IDD. METHODS: We assessed: BMI (kg/m2), moderate-to-vigorous physical activity (MVPA), fruit and vegetable intake (FVI), parental diet and physical activity habits, and socioeconomic characteristics. Associations of BMI on MVPA and FVI were assessed with Spearman's correlation; differences in BMI by parental diet and physical activity habits were assessed with Kruskall-Wallis tests; and the relationships of BMI to household income, race, and education were assessed with Kendall Tau-b and Mann Whitney U tests. RESULTS: Data was obtained from 110 parents (97.3% female) who were study partners for their adolescents/young adults with IDD participating in a weight loss clinical trial. Approximately 81% of parents were overweight or obese (25.7% overweight, 55.1% obese), with 46.3% and 20% meeting the recommended U.S. guidelines for MVPA and FVI, respectively. Higher FVI and higher income were significantly associated with lower parent BMI. BMI was significantly lower in parents who reported to be physically active and choose healthy food. CONCLUSION: We observed a high prevalence of overweight/obesity, low FVI and low levels of MVPA in parents of adolescents with IDD. These observations suggest that interventions designed to address these factors have the potential to improve the health and wellbeing of both parents and adolescents with IDD. CLINICAL TRIALS NUMBER: NCT02561754.

Familiarity and Use of MyPlate: An Online Focus Group Exploration Among Midwestern K-12 Teachers.

OBJECTIVE: To examine teachers' familiarity and use of MyPlate, including barriers to using it. METHODS: Twenty kindergarten through grade 12 teachers were recruited from 1 urban and suburban school district in the Midwest to participate in virtual focus groups regarding familiarity, use, and barriers to MyPlate. A basic descriptive qualitative approach with thematic analysis was guided by systems thinking. Common categories were coded and agreed on by the authors. RESULTS: Findings included main categories of individual awareness, use in curriculum, and appropriate facilitators of MyPlate. Awareness and use of MyPlate were mixed. Teachers integrated MyPlate in math, history, and other subjects. Barriers included packed curriculum and cultural issues. The facilitators of MyPlate mentioned were health or physical education teachers. CONCLUSIONS AND IMPLICATIONS: Online focus groups successfully collected formative data on teachers' perspectives toward MyPlate. The technology could be used in future similar research. Enhanced teacher training may improve the integration of MyPlate into schools. School teachers identified major barriers to MyPlate in the classroom, including lack of time and resources. There was mixed feedback on how MyPlate and nutrition may be used in school curricula. Enhanced teacher training may improve the integration of MyPlate into schools.

Weight loss in adolescents with down syndrome compared to adolescents with other intellectual disabilities enrolled in an 18-month randomized weight management trial.

Background: There is limited information on the efficacy of weight management interventions in adolescents with Down Syndrome (DS). Objective: To compare weight change and intervention compliance between adolescents with DS compared to adolescents with non-DS related intellectual disabilities (ID) who were enrolled in an 18-month weight management trial. Methods: Participants were adolescents (13-21 years) with mild to moderate ID and overweight or obesity. Participants were randomized in a 1:1:1 allocation to one of 3 intervention arms for an 18-month weight management trial: face-to-face/conventional diet (FTF/CD), remote delivery/conventional diet (RD/CD), or remote delivery/enhanced Stop Light Diet (RD/eSLD). Anthropometrics were assessed at baseline 6, 12, and 18 months by staff blinded to the intervention, and self-monitoring data was collected across the 18-month study. As an unpowered, post-hoc, secondary analysis, two-sample t-tests were used to compare the weight change across 6,12, and 18 mos. and compliance across 18 mos. between adolescents with and without DS randomized to each intervention arm. Results: Adolescents with ID (n = 110) were randomized to one of three intervention arms: FTF/CD (n = 36, DS = 17, other ID = 19), RD/CD (n = 39, DS = 21, other ID = 18) or RD/eSLD (n = 35, DS = 15, other ID = 20). Body weight at 18 months was obtained from 82%, 76% and 73% of participants with DS and 84%, 83% and 75% of participants with other ID randomized to the FTF/CD, RD/CD, and RD/eSLD arms, respectively Weight change across 18 months was -0.2 ± 8.8 kg (-0.5%), -0.3 ± 5.3 kg (-0.7%), and -2.6 ± 5.0 kg (-4.0%) in adolescents with DS randomized to the FTF/CD, RD/CD and RD/eSLD arms, respectively. There were no significant differences in change in body weight or BMI across 18 months between adolescents with DS or those with other ID in any of the 3 intervention arms (all p > 0.05). Additionally, there were no significant differences in intervention compliance between adolescents with and without DS across 18 mos. (all p > 0.05). Conclusions: Adolescents with DS respond to a multi-component weight management intervention similar to those with others ID.

Host defense functions of the epididymal amyloid matrix.

The epididymal lumen is an immunologically distinct environment. It maintains tolerance for the naturally antigenic spermatozoa to allow their maturation into functional cells while simultaneously defending against pathogens that can ascend the male tract and cause infertility. We previously demonstrated that a nonpathological amyloid matrix that includes several cystatin-related epididymal spermatogenic (CRES) subgroup family members is distributed throughout the mouse epididymal lumen but its function was unknown. Here, we reveal a role for the epididymal amyloid matrix in host defense and demonstrate that the CRES amyloids and CD-1 mouse epididymal amyloid matrix exhibit potent antimicrobial activity against bacterial strains that commonly cause epididymal infections in men. We show the CRES and epididymal amyloids use several defense mechanisms including bacterial trapping, disruption of bacterial membranes and promotion of unique bacterial ghost-like structures. Remarkably, these antimicrobial actions varied depending on the bacterial strain indicating CRES amyloids and the epididymal amyloids elicit strain-specific host defense responses. We also demonstrate that the CRES monomer and immature assemblies of the epididymal amyloid transitioned into advanced structures in the presence of bacteria, suggesting their amyloid-forming/shape-shifting properties allows for a rapid reaction to a pathogen and provides an inherent plasticity in their host defense response. Together, our studies reveal new mechanistic insight into how the male reproductive tract defends against pathogens. Future studies using a mouse model for human epididymitis are needed to establish the epididymal amyloid responses to pathogens in vivo. Broadly, our studies provide an example of why nature has maintained the amyloid fold throughout evolution.

Feasibility and Comparative Effectiveness for the Delivery of the National Diabetes Prevention Program through Cooperative Extension in Rural Communities.

The U.S. Cooperative Extension Service (CE) has potential to deliver the National Diabetes Prevention Program (NDPP) to rural residents with prediabetes. However, the CE remains underutilized for the delivery of NDPP. We compared the feasibility/effectiveness of the NDPP (0-6 mos.) delivered by CE personnel to rural residents with prediabetes using Zoom® (CE-Zoom®) or by our research staff using Facebook® (FB). Adults (n = 31, age ~55 years) were enrolled (CE-Zoom® n = 16, FB n = 15). Attendance did not differ significantly between groups (CE Zoom® = 69%, FB = 83%, p = 0.15). Participant retention was similar in the CE Zoom® (88%) and FB groups (87%). CE-Zoom® and FB® groups provided weekly, self-monitoring data for 83% and 84% of the 24 potential weeks, respectively. Six-month weight loss was not different between groups (CE-Zoom® = -5.99 ± 8.0 kg, -5.4%, FB = -1.68 ± 3.3 kg, -1.6% p = 0.13). Participants achieving ≥5% weight loss was greater in the CE-Zoom® (44%) compared with the FB group (7%, p = 0.04). Participants achieving the NDPP program goal for physical activity (≥150 min/week) did not differ (CE-Zoom® = 75%, FB = 67%, p = 0.91). This pilot trial demonstrated the potential feasibility and effectiveness of the NDPP delivered by CE personnel in a group remote format (Zoom®) to adults with prediabetes living in rural areas.

The impact of exercise and cumulative physical activity on energy intake and diet quality in adults enrolled in the Midwest Exercise Trial for the Prevention of Weight Regain.

The purpose of this study was to assess impact of different volumes of exercise as well as cumulative moderate to vigorous physical activity (MVPA) on energy intake (EI) and diet quality, as assessed by the Healthy Eating Index-2010(HEI-2010), across a 12-month weight maintenance intervention. Participants were asked to attend group behavioural sessions, eat a diet designed for weight maintenance and exercise either 150, 225 or 300 min/week. Dietary intake was assessed by 3-d food records, and MVPA was assessed by accelerometry. Two hundred and twenty-four participants (42·5 years of age, 82 % female) provided valid dietary data for at least one time point. There was no evidence of group differences in EI, total HEI-2010 score or any of the HEI-2010 component scores (all P > 0·05). After adjusting for age, sex, time, group and group-by-time interactions, there was an effect of cumulative MVPA on EI (1·08, P = 0·04), total HEI-2010 scores (-0·02, P = 0·003), Na (-0·006, P = 0·002) and empty energy scores (-0·007, P = 0·004. There was evidence of a small relationship between cumulative daily EI and weight (ß: 0·00187, 95 % CI 0·001, P = 0·003). However, there was no evidence for a relationship between HEI total score (ß: -0·006, 95 % CI 0·07, 0·06) or component scores (all P > 0·05) and change in weight across time. The results of this study suggest that increased cumulative MVPA is associated with clinically insignificant increases in EI and decreases in HEI.

High-throughput imaging of fresh-frozen plant reproductive samples in a variable pressure SEM.

Conventional light and electron microscopy are the most widely used techniques for examining plant reproductive tissues; however, they are time-consuming or expensive. The anther is the male part of the plant reproductive system. Structural changes drive development, and any structural defect may lead to an increase in fertility or cause sterility; thus, quick detection of structural changes is crucial in reproductive biology. We optimized an existing low-temperature SEM alternative to examine the internal structure of hydrated, fresh-frozen anthers. In contrast with the original technique, our method does not require precooling adhesion (ethanol to fix the specimen), and the cryo-sectioning can be conducted at atmospheric pressure. In addition to enabling the differentiation between aerial and liquid-filled intercellular spaces, this method is expected to facilitate the detection of quick (during a day) developmental changes in plant reproductive tissues, which is a current challenge using conventional approaches.•This method allows the high-throughput imaging of fresh-frozen plant reproductive samples collected every 10 min, which is important for developmental studies.•The cryo-images of samples with thickness ranging from 0.2 to 3 mm can be well-preserved at 800X magnification.•This method does not require chemical processing, critical point drying, customized cryo-accessories, controlled temperature cold stages, or metal coating. This simplified method does not require highly skilled personnel, and it is suitable in most microscopy laboratories.

Calcium, fiber, iron, and sodium intake in adolescents with intellectual and developmental disabilities and overweight and obesity.

BACKGROUND: Adolescents with intellectual and developmental disabilities (IDD) and overweight or obesity (OW/OB) are a nutritionally vulnerable group with increased risk of nutritional deficiencies. However, there are limited data examining micronutrient intake in adolescents with IDD and OW/OB. OBJECTIVE: The purpose of this study was to assess the adequacy of calcium, iron, fiber, and sodium intake referenced against the United States Dietary Reference Intakes in adolescents with IDD and OW/OB. METHODS: Three-day image-assisted food records were used to assess dietary intake of 64 adolescents with IDD and OW/OB. A mean ± standard deviation was calculated for mean intake of calcium (mg), fiber (g/1000 kcals energy), iron (mg), and sodium (mg). RESULTS: A total of 157 nutrient intake observations were completed by 64 participants (56% female, 16.3 ± 2.3 years). Calcium intake for participants ages 14-18 years (n = 57) was 1027.4 ± 607.5 mg, which is below the EAR of 1050 mg. Calcium intake for participants ages ≥19 years (n = 7) was 921.1 ± 596.4 mg, which is greater than the EAR of 840 mg. Fiber intake was 8.4 ± 3.6 g/1000 kcals, which is below the AI of 14 g/1000 kcals. Iron intake for all participants exceeded their respective EARs. Sodium intake was 3180.9 ± 975.9 mg, which above the AI of 2300 mg. CONCLUSION: Calcium intake was adequate for participants ≥19 years of age, but inadequate for participants 14-18 years. For all participants, iron and sodium intake exceeded the DRI while fiber intake was below the DRI.

Cross-seeding between the functional amyloidogenic CRES and CRES3 family members and their regulation of Aß assembly.

Accumulating evidence shows that amyloids perform biological roles. We previously showed that an amyloid matrix composed of four members of the CRES subgroup of reproductive family 2 cystatins is a normal component of the mouse epididymal lumen. The cellular mechanisms that control the assembly of these and other functional amyloid structures, however, remain unclear. We speculated that cross-seeding between CRES members could be a mechanism to control the assembly of the endogenous functional amyloid. Herein we used thioflavin T assays and negative stain transmission electron microscopy to explore this possibility. We show that CRES3 rapidly formed large networks of beaded chains that possessed the characteristic cross-ß reflections of amyloid when examined by X-ray diffraction. The beaded amyloids accelerated the amyloidogenesis of CRES, a less amyloidogenic family member, in seeding assays during which beads transitioned into films and fibrils. Similarly, CRES seeds expedited CRES3 amyloidogenesis, although less efficiently than the CRES3 seeding of CRES. These studies suggest that CRES and CRES3 hetero-oligomerize and that CRES3 beaded amyloids may function as stable preassembled seeds. The CRES3 beaded amyloids also facilitated assembly of the unrelated amyloidogenic precursor Aß by providing a surface for polymerization though, intriguingly, CRES3 (and CRES) monomer/early oligomer profoundly inhibited Aß assembly. The cross-seeding between the CRES subgroup members is similar to that which occurs between bacterial curli proteins suggesting that it may be an evolutionarily conserved mechanism to control the assembly of some functional amyloids. Further, interactions between unrelated amyloidogenic precursors may also be a means to regulate functional amyloid assembly.

The Functional Mammalian CRES (Cystatin-Related Epididymal Spermatogenic) Amyloid is Antiparallel ß-Sheet Rich and Forms a Metastable Oligomer During Assembly.

An amyloid matrix composed of several family 2 cystatins, including the reproductive cystatin CRES, is an integral structure in the mouse epididymal lumen and has proposed functions in sperm maturation and protection. Understanding how CRES amyloid assembles in vitro may provide clues on how the epididymal amyloid matrix forms in vivo. We therefore purified full-length CRES under nondenaturing conditions and followed its aggregation from monomer to amyloid under conditions that may approximate those in the epididymal lumen. CRES transitioned into a metastable oligomer that was resistant to aggregation and only over extended time formed higher-ordered amyloids. High protein concentrations facilitated oligomer assembly and also were required to maintain the metastable state since following dilution the oligomer was no longer detected. Similar to other amyloid precursors, the formation of CRES amyloids correlated with a loss of α-helix and a gain of ß-sheet content. However, CRES is unique in that its amyloids are rich in antiparallel ß-sheets instead of the more common parallel ß-sheets. Taken together, our studies suggest that early metastable oligomers may serve as building blocks for functional amyloid assembly and further reveal that antiparallel ß-sheet-rich amyloids can be functional forms.

Cystatin-related epididymal spermatogenic subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen.

STUDY QUESTION: Do the CRES (cystatin-related epididymal spermatogenic) subgroup members, including CRES2, CRES3 and cystatin E2, contribute to the formation of a nonpathological, functional amyloid matrix in the mouse epididymal lumen? SUMMARY ANSWER: CRES2, CRES3 and cystatin E2 self-assemble with different aggregation properties into amyloids in vitro, are part of a common amyloid matrix in the mouse epididymal lumen and are present in extracellular vesicles. WHAT IS KNOWN ALREADY: Although previously thought only to be pathological, accumulating evidence has established that amyloids, which are highly ordered protein aggregates, can also carry out functional roles in the absence of pathology. We previously demonstrated that nonpathological amyloids are present in the epididymis; specifically, that the reproductive cystatin CRES forms amyloid and is present in the mouse epididymal lumen in a film-like amyloid matrix that is intimately associated with spermatozoa. Because the related proteins CRES2, CRES3 and cystatin E2 are also expressed in the epididymis, the present studies were carried out to determine if these proteins are also amyloidogenic in vitro and in vivo and thus may coordinately function with CRES as an amyloid structure. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: The epididymides from CD1 and Cst8 (CRES)129SvEv/B6 gene knockout (KO) and wild-type mice and antibodies that specifically recognize each CRES subgroup member were used for immunohistochemical and biochemical analyzes of CRES subgroup proteins. Methods classically used to identify amyloid, including the conformation-dependent dyes thioflavin S (ThS) and thioflavin T (ThT), conformation-dependent antibodies, protein aggregation disease ligand (which binds any amyloid independent of sequence) and negative stain electron microscopy (EM) were carried out to examine the amyloidogenic properties of CRES subgroup members. Immunofluorescence analysis and confocal microscopy were used for colocalization studies. MAIN RESULTS AND THE ROLE OF CHANCE: Immunoblot and immunofluorescence analyzes showed that CRES2, CRES3 and cystatin E2 were primarily found in the initial segment and intermediate zone of the epididymis and were profoundly downregulated in epididymides from CRES KO mice, suggesting integrated functions. Except for CRES3, which was only detected in a particulate form, proteins were present in the epididymal lumen in both soluble and particulate forms including in a film-like matrix and in extracellular vesicles. The use of amyloid-specific reagents determined that all CRES subgroup members were present as amyloids and colocalized to a common amyloid matrix present in the epididymal lumen. Negative stain EM, dot blot analysis and ThT plate assays showed that recombinant CRES2, CRES3 and cystatin E2 formed amyloid in vitro, albeit with different aggregation properties. Together, our studies demonstrate that a unique amyloid matrix composed of the CRES family of reproductive-specific cystatins and cystatin C is a normal component of the mouse epididymal lumen and may play a functional role in sperm maturation by coordinating interactions between the luminal fluid and spermatozoa. LIMITATIONS, REASONS FOR CAUTION: The structures examined in our studies were isolated from luminal fluid obtained by puncture of the epididymis and therefore we cannot rule out some contamination by epithelial cells. Although our studies show CRES family members are associated with extracellular vesicles, we have yet to determine if proteins are present on the surface or are within the vesicles. We also have not established if narrow/apical cells are the source of the CRES family extracellular vesicles. CRES and CRES2 have been previously found in the human epididymis and associated with spermatozoa; however, we have yet to determine if the human CRES subgroup proteins are amyloidogenic and if an amyloid matrix is present in the human epididymal lumen. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the regulation and biological roles of amyloids, such as the CRES subgroup amyloid matrix that functions without causing pathology, could have broad implications for understanding pathological amyloids including those associated with neurodegenerative diseases and prionopathies. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by NIH grants RO1HD033903 and RO1HD056182 to G.A.C. The authors declare there are no conflicts of interest.

Drak Is Required for Actomyosin Organization During Drosophila Cellularization.

The generation of force by actomyosin contraction is critical for a variety of cellular and developmental processes. Nonmuscle myosin II is the motor that drives actomyosin contraction, and its activity is largely regulated by phosphorylation of the myosin regulatory light chain. During the formation of the Drosophila cellular blastoderm, actomyosin contraction drives constriction of microfilament rings, modified cytokinesis rings. Here, we find that Drak is necessary for most of the phosphorylation of the myosin regulatory light chain during cellularization. We show that Drak is required for organization of myosin II within the microfilament rings. Proper actomyosin contraction of the microfilament rings during cellularization also requires Drak activity. Constitutive activation of myosin regulatory light chain bypasses the requirement for Drak, suggesting that actomyosin organization and contraction are mediated through Drak's regulation of myosin activity. Drak is also involved in the maintenance of furrow canal structure and lateral plasma membrane integrity during cellularization. Together, our observations suggest that Drak is the primary regulator of actomyosin dynamics during cellularization.

Nonpathological extracellular amyloid is present during normal epididymal sperm maturation.

Amyloids are aggregated proteins characterized by a specific cross-ß-sheet structure and are typically associated with neurodegenerative diseases including Alzheimer's disease. Recently, however, several nonpathological amyloids have been found in intracellular organelles of normal mammalian tissues suggesting that amyloid may also carry out biological functions. We previously have shown that the epididymal cystatin CRES (cystatin-related epididymal spermatogenic), cst8, a reproductive-specific member of the cystatin superfamily of cysteine protease inhibitors, forms amyloid in vitro suggesting that CRES amyloid may also form in vivo within the epididymal lumen. Here we show that amyloid structures containing CRES are a component of the normal mouse epididymal lumen without any apparent cytotoxic effects on spermatozoa and that these structures change along the length of the tubule. These studies suggest the presence of a functional amyloid structure that may carry out roles in sperm maturation or maintenance of the luminal milieu and which itself may undergo maturational changes along the epididymis. In contrast to previous examples of functional amyloid which were intracellular, our studies now show that nonpathological/functional amyloid can also be extracellular. The presence of an extracellular and nonpathological amyloid in the epididymis suggests that similar amyloid structures may be present in other organ systems and may carry out distinctive tissue-specific functions.

Oligomerization and transglutaminase cross-linking of the cystatin CRES in the mouse epididymal lumen: potential mechanism of extracellular quality control.

CRES (cystatin-related epididymal spermatogenic), a member of the cystatin superfamily of cysteine protease inhibitors, is expressed in the epididymis and spermatozoa, suggesting specialized roles in reproduction. Several cystatin family members oligomerize, including cystatin C that forms amyloid deposits associated with cerebral amyloid angiopathy. Our studies demonstrate that CRES also forms oligomers. Size exclusion chromatography revealed the presence of multiple forms of CRES in the epididymal luminal fluid, including SDS-sensitive and SDS-resistant high molecular mass complexes. In vitro experiments demonstrated that CRES is a substrate for transglutaminase and that an endogenous transglutaminase activity in the epididymal lumen catalyzed the formation of SDS-resistant CRES complexes. The use of a conformation-dependent antibody that recognizes only the oligomeric precursors to amyloid, negative stain electron microscopy, and Congo Red staining showed that CRES adopted similar oligomeric and fibrillar structures during its aggregation as other amyloidogenic proteins, suggesting that CRES has the potential to form amyloid in the epididymal lumen. The addition of transglutaminase, however, prevented the formation of CRES oligomers recognized by the conformation antibody by cross-linking CRES into an amorphous structure. We propose that transglutaminase activity in the epididymal lumen may function as a mechanism of extracellular quality control by diverting proteins such as CRES from the amyloidogenic pathway.

Pseudomonas aeruginosa forms biofilms in acute infection independent of cell-to-cell signaling.

Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.