RESUMEN
ABSTRACT: Scott, KM, Kreisel, BR, Florkiewicz, EM, Crowell, MS, Morris, JB, McHenry, PA, and Benedict, TM. The effect of cautionary versus resiliency spine education on maximum deadlift performance and back beliefs: A randomized control trial. J Strength Cond Res 38(7): e341-e348, 2024-The purpose of this study was to determine the effect of cautionary information about the spine vs. a message of spine resiliency on maximum deadlift (MDL) performance and beliefs regarding the vulnerability of the spine. This cluster randomized control trial involved 903 military new cadets (n = 903) during their mandatory fitness test in cadet basic training (mean age 18.3 years, body mass index 23.8 kg·m-2, 22% female). Subjects were cluster randomized to 3 groups. The cautionary group received a message warning them to protect their backs while deadlifting, the resiliency group received a message encouraging confidence while deadlifting, and the control group received the standardized Army deadlift education only. The outcome measures were MDL weight lifted and perceived spine vulnerability. Significance was set at alpha ≤0.05. There were no between-group differences in weight lifted (p=0.40). Most subjects believed that the spine is vulnerable to injury. Three times as many subjects who received the resiliency education improved their beliefs about the vulnerability of their spines compared with those receiving the cautionary education (p<0.001). This study demonstrated the potential for brief resiliency education to positively influence beliefs about spine vulnerability, whereas cautionary education did not impair performance.
Asunto(s)
Personal Militar , Humanos , Femenino , Masculino , Personal Militar/psicología , Adolescente , Adulto Joven , Levantamiento de Peso/fisiología , Levantamiento de Peso/psicología , Traumatismos de la Espalda/prevención & control , Columna Vertebral/fisiología , AdultoRESUMEN
Plasma levels of HDL cholesterol are inversely associated with CVD progression. It is becoming increasingly clear that HDL plays important roles in immunity that go beyond its traditionally understood roles in lipid transport. We previously reported that HDL interaction with regulatory T cells (Treg) protected them from apoptosis, which could be a mechanism underlying the broad anti-inflammatory effect of HDL. Herein, we extend our work to show that HDL interacts mainly with memory Treg, particularly with the highly suppressive effector memory Treg, by limiting caspase-dependent apoptosis in an Akt-dependent manner. Reconstitution experiments identified the protein component of HDL as the primary driver of the effect, though the most abundant HDL protein, apolipoprotein A-I (APOA1), was inactive. In contrast, APOE-depleted HDL failed to rescue effector memory Treg, suggesting the critical role of APOE proteins. HDL particles reconstituted with APOE, and synthetic phospholipids blunted Treg apoptosis at physiological concentrations. The APOE3 and APOE4 isoforms were the most efficient. Similar results were obtained when lipid-free recombinant APOEs were tested. Binding experiments showed that lipid-free APOE3 bound to memory Treg but not to naive Treg. Overall, our results show that APOE interaction with Treg results in blunted caspase-dependent apoptosis and increased survival. As dysregulation of HDL-APOE levels has been reported in CVD and obesity, our data bring new insight on how this defect may contribute to these diseases.
Asunto(s)
Enfermedades Cardiovasculares , Linfocitos T Reguladores , Humanos , Linfocitos T Reguladores/metabolismo , Apolipoproteína E3/metabolismo , Apolipoproteínas E/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismoRESUMEN
High levels of circulating triglycerides (TGs), or hypertriglyceridemia, are key components of metabolic diseases, such as type 2 diabetes, metabolic syndrome, and CVD. As TGs are carried by lipoproteins in plasma, hypertriglyceridemia can result from overproduction or lack of clearance of TG-rich lipoproteins (TRLs) such as VLDLs. The primary driver of TRL clearance is TG hydrolysis mediated by LPL. LPL is regulated by numerous TRL protein components, including the cofactor apolipoprotein C-II, but it is not clear how their effects combine to impact TRL hydrolysis across individuals. Using a novel assay designed to mimic human plasma conditions in vitro, we tested the ability of VLDL from 15 normolipidemic donors to act as substrates for human LPL. We found a striking 10-fold difference in hydrolysis rates across individuals when the particles were compared on a protein or a TG basis. While VLDL TG contents moderately correlated with hydrolysis rate, we noticed substantial variations in non-apoB proteins within these particles by MS. The ability of LPL to hydrolyze VLDL TGs did not correlate with apolipoprotein C-II content, but it was strongly inversely correlated with apolipoprotein E (APOE) and, to a lesser extent, apolipoprotein A-II. Addition of exogenous APOE inhibited LPL lipolysis in a dose-dependent manner. The APOE3 and (particularly) APOE4 isoforms were effective at limiting LPL hydrolysis, whereas APOE2 was not. We conclude that APOE on VLDL modulates LPL activity and could be a relevant factor in the pathogenesis of metabolic disease.
Asunto(s)
Diabetes Mellitus Tipo 2RESUMEN
Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1's ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the "double super helix." Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.
Asunto(s)
Apolipoproteína A-I , Colesterol , Transportador 1 de Casete de Unión a ATP/metabolismo , Apolipoproteína A-I/metabolismo , Transporte Biológico , Colesterol/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Fosfolípidos/metabolismoRESUMEN
Pregnancy is accompanied by significant physiological changes, which can impact the health and development of the fetus and mother. Pregnancy-induced changes in plasma lipoproteins are well documented, with modest to no impact observed on the generic measure of high density lipoprotein (HDL) cholesterol. However, the impact of pregnancy on the concentration and composition of HDL subspecies has not been examined in depth. In this prospective study, we collected plasma from 24 nonpregnant and 19 pregnant women in their second trimester. Using nuclear magnetic resonance (NMR), we quantified 11 different lipoprotein subspecies from plasma by size, including three in the HDL class. We observed an increase in the number of larger HDL particles in pregnant women, which were confirmed by tracking phospholipids across lipoproteins using high-resolution gel-filtration chromatography. Using liquid chromatography-mass spectrometry (LC-MS), we identified 87 lipid-associated proteins across size-speciated fractions. We report drastic shifts in multiple protein clusters across different HDL size fractions in pregnant females compared with nonpregnant controls that have major implications on HDL function. These findings significantly elevate our understanding of how changes in lipoprotein metabolism during pregnancy could impact the health of both the fetus and the mother.
Asunto(s)
Lipoproteínas HDL/química , Adolescente , Adulto , Cromatografía Liquida , Femenino , Humanos , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Tamaño de la Partícula , Proteoma/química , Adulto JovenRESUMEN
The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein crosslinker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation product-mediated crosslinking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well-studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ± 33.8 pmol/mg) than healthy controls (15.3 ± 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq), which was 100-fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from â¼46.5 to â¼18.4% (p < 0.001). Surprisingly, ONE modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC-MS/MS analysis revealed that Lys-12, Lys-23, Lys-96, and Lys-226 in apoA-I are modified by ONE ketoamide adducts. Compared with other dicarbonyl scavengers, pentylpyridoxamine (PPM) most efficaciously blocked ONE-induced protein crosslinking in HDL and also prevented HDL dysfunction in an in vitro model of inflammation. Our findings show that ONE-HDL adducts cause HDL dysfunction and are elevated in individuals with FH who have severe hypercholesterolemia.
Asunto(s)
Aldehídos/metabolismo , Hiperlipoproteinemia Tipo II/metabolismo , Lipoproteínas HDL/metabolismo , Lisina/metabolismo , Aldehídos/análisis , Animales , Apolipoproteína A-I/metabolismo , Aterosclerosis/metabolismo , Células Cultivadas , Femenino , Humanos , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/diagnóstico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity (P < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.
Asunto(s)
Apolipoproteína A-I/metabolismo , HDL-Colesterol/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Apolipoproteína A-I/genética , Activación Enzimática , Humanos , MutaciónRESUMEN
Apolipoprotein (apo) A-I mediates many of the anti-atherogenic functions attributed to high density lipoprotein. Unfortunately, efforts toward a high resolution structure of full-length apoA-I have not been fruitful, although there have been successes with deletion mutants. Recently, a C-terminal truncation (apoA-I(Δ185-243)) was crystallized as a dimer. The structure showed two helical bundles connected by a long, curved pair of swapped helical domains. To compare this structure to that existing under solution conditions, we applied small angle x-ray scattering and isotope-assisted chemical cross-linking to apoA-I(Δ185-243) in its dimeric and monomeric forms. For the dimer, we found evidence for the shared domains and aspects of the N-terminal bundles, but not the molecular curvature seen in the crystal. We also found that the N-terminal bundles equilibrate between open and closed states. Interestingly, this movement is one of the transitions proposed during lipid binding. The monomer was consistent with a model in which the long shared helix doubles back onto the helical bundle. Combined with the crystal structure, these data offer an important starting point to understand the molecular details of high density lipoprotein biogenesis.
Asunto(s)
Apolipoproteína A-I/química , Metabolismo de los Lípidos , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Unión Proteica , Multimerización de ProteínaRESUMEN
Apolipoprotein (apo)A-IV is a lipid emulsifying protein linked to a range of protective roles in obesity, diabetes, and cardiovascular disease. It exists in several states in plasma including lipid-bound in HDL and chylomicrons and as monomeric and dimeric lipid-free/poor forms. Our recent x-ray crystal structure of the central domain of apoA-IV shows that it adopts an elongated helical structure that dimerizes via two long reciprocating helices. A striking feature is the alignment of conserved proline residues across the dimer interface. We speculated that this plays important roles in the structure of the lipid-free protein and its ability to bind lipid. Here we show that the systematic conversion of these prolines to alanine increased the thermodynamic stability of apoA-IV and its propensity to oligomerize. Despite the structural stabilization, we noted an increase in the ability to bind and reorganize lipids and to promote cholesterol efflux from cells. The novel properties of these mutants allowed us to isolate the first trimeric form of an exchangeable apolipoprotein and characterize it by small-angle x-ray scattering and chemical cross-linking. The results suggest that the reciprocating helix interaction is a common feature of all apoA-IV oligomers. We propose a model of how self-association of apoA-IV can result in spherical lipoprotein particles, a model that may have broader applications to other exchangeable apolipoprotein family members.
Asunto(s)
Apolipoproteínas A/química , Apolipoproteínas A/metabolismo , Alanina/química , Sustitución de Aminoácidos , Apolipoproteínas A/genética , Colesterol/metabolismo , Secuencia Conservada , Humanos , Liposomas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Prolina/química , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Termodinámica , Difracción de Rayos XRESUMEN
Apolipoprotein (apo)A-IV plays important roles in dietary lipid and glucose metabolism, and knowledge of its structure is required to fully understand the molecular basis of these functions. However, typical of the entire class of exchangeable apolipoproteins, its dynamic nature and affinity for lipid has posed challenges to traditional high resolution structural approaches. We previously reported an x-ray crystal structure of a dimeric truncation mutant of apoA-IV, which showed a unique helix-swapping molecular interface. Unfortunately, the structures of the N and C termini that are important for lipid binding were not visualized. To build a more complete model, we used chemical cross-linking to derive distance constraints across the full-length protein. The approach was enhanced with stable isotope labeling to overcome ambiguities in determining molecular span of the cross-links given the remarkable similarities in the monomeric and dimeric apoA-IV structures. Using 51 distance constraints, we created a starting model for full-length monomeric apoA-IV and then subjected it to two modeling approaches: (i) molecular dynamics simulations and (ii) fitting to small angle x-ray scattering data. This resulted in the most detailed models yet for lipid-free monomeric or dimeric apoA-IV. Importantly, these models were of sufficient detail to direct the experimental identification of new functional residues that participate in a "clasp" mechanism to modulate apoA-IV lipid affinity. The isotope-assisted cross-linking approach should prove useful for further study of this family of apolipoproteins in both the lipid-free and -bound states.
Asunto(s)
Apolipoproteínas A/química , Simulación de Dinámica Molecular , Apolipoproteínas A/genética , Cristalografía por Rayos X , Humanos , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
ApoA-IV is an amphipathic protein that can emulsify lipids and has been linked to protective roles against cardiovascular disease and obesity. We previously reported an x-ray crystal structure of apoA-IV that was truncated at its N and C termini. Here, we have extended this work by demonstrating that self-associated states of apoA-IV are stable and can be structurally studied using small-angle x-ray scattering. Both the full-length monomeric and dimeric forms of apoA-IV were examined, with the dimer showing an elongated rod core with two nodes at opposing ends. The monomer is roughly half the length of the dimer with a single node. Small-angle x-ray scattering visualization of several deletion mutants revealed that removal of both termini can have substantial conformational effects throughout the molecule. Additionally, the F334A point mutation, which we previously showed increases apoA-IV lipid binding, also exhibited large conformational effects on the entire dimer. Merging this study's low-resolution structural information with the crystal structure provides insight on the conformation of apoA-IV as a monomer and as a dimer and further defines that a clasp mechanism may control lipid binding and, ultimately, protein function.
Asunto(s)
Apolipoproteínas A/química , Apolipoproteínas A/metabolismo , Biofisica/métodos , Dimerización , Dimiristoilfosfatidilcolina/química , Humanos , Lípidos/química , Conformación Molecular , Mutación Puntual , Conformación Proteica , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Ultracentrifugación , Rayos XRESUMEN
Background: Limitations exist with current ACLR functional testing assessments that may be mitigated by including single-leg multi-directional testing. Hypothesis/Purpose: To compare Anterior Cruciate Ligament-Return to Sport after Injury Scale (ACL-RSI) scores, limb symmetry index (LSI) of the single-leg vertical jump (SLVJ), single-leg medial rotation hop (SLMRH), single-leg lateral hop (SLLH), and isokinetic quadriceps strength between participants with an ACLR and healthy controls and assess predictors of quadriceps strength asymmetry. It was hypothesized that ACL-RSI scores and LSIs for all tests would differ between ACLR and healthy control groups and within the ACLR group a strong correlation would exist between all outcome measures and quadriceps strength. Study Design: Cross-Sectional Study. Methods: Twenty-six participants with an ACLR (median 13 months after surgery) and twenty-six matched healthy controls were recruited to participate in this study. Performance was assessed via SLVJ, SLMRH, SLLH, and isokinetic quadriceps strength. Between-group comparisons were made with independent t-tests and Mann-Whitney U test. Within the ACLR group, bivariate correlation and multivariate regression analysis were performed to assess the relationship between the outcome measures and quadriceps strength asymmetry. Results: Significant between-limb differences were only identified in the ACLR group (p< 0.05): SLVJ LSI: 88.5%, SLMRH LSI: 93.6%, SLLH LSI: 92.7%, quadriceps strength LSI 80.9% - 83.9%, which were significantly lower (p <0.05) than the healthy control group. Within the ACLR group, a moderate-strong significant (p < 0.05) correlations existed with quadriceps strength and SLVJ (r=0.44-0.65), SLMRH (r =0.43-0.83), and SLLH (r=0.54-0.63); while ACL-RSI had a weak non-significant (p > 0.05) correlation with quadriceps strength (r= 0.12-0.30). Conclusion: Single-leg multidirectional test LSIs were less in ACLR participants than matched healthy controls and all were directly related to quadriceps strength. Psychological readiness to return to sport was not related to quadriceps strength.
RESUMEN
BACKGROUND: Traumatic shoulder instability is a common injury in the general population and the military. Surgical stabilization surgery reduces recurrence rates compared with nonsurgical management. Time since surgery is generally the primary measure of return to sport. There is a gap in knowledge on psychological variables and time since surgery and their relationship to rotator cuff strength and functional performance. HYPOTHESIS: It was hypothesized that, after shoulder stabilization surgery, psychological factors and time since surgery will be associated positively with objective physical performance tests, that physical performance will differ significantly between postsurgery cadets and healthy controls, and that surgical stabilization of the nondominant arm will demonstrate greater range of motion deficits than surgical intervention on the dominant arm. STUDY DESIGN: Case-control study. LEVEL OF EVIDENCE: Level 4. METHODS: The 52 participants (26 postsurgical [6-24 months after surgery] and 26 healthy controls) were all military cadets. Outcome measures were patient-reported outcomes, range of motion, isometric strength, and functional performance. RESULTS: No significant relationships existed between time since surgery and psychological factors to rotator cuff strength or functional performance. Significant differences were found between groups in self-reported outcomes, including the Shoulder Instability Return to Sport After Injury scale, Single Assessment Numeric Evaluation, Numeric Pain Rating Scale, quickDASH, flexion and external rotation (ER), and ER limb symmetry. Those who received dominant-sided shoulder surgery demonstrated a greater mean active range of motion deficit than those who received nondominant-sided surgery. Both groups demonstrated a significant loss in ER, but dominant-sided surgical participants also demonstrated significant flexion loss. CONCLUSION: Time since surgery and psychological variables did not demonstrate a relationship to rotator cuff strength and functional performance. Significant differences existed between the stabilization surgical participants and healthy controls in all patient-reported outcomes. Surgical participants with dominant-sided shoulder surgery demonstrated a greater mean motion deficit when compared with those who received nondominant-sided surgery.
RESUMEN
Background: Concussions are often accompanied by balance disturbances. Clinically accurate evaluation systems are often expensive, large, and inaccessible to most clinicians. The Sway Balance Mobile Application (SWAY) is an accessible method to quantify balance changes. Purpose: To determine the known groups and convergent validity of the SWAY to assess balance after a concussion. Study Design: Case-Control Study. Methods: Twenty participants with acute concussion and twenty controls were recruited. At initial, one-week, and final return to activity (RTA) evaluations, all participants completed the Sports Concussion Assessment Tool (SCAT-5), and balance control measured by SWAY mBESS and NeuroCom Balance Master Sensory Organization Test (SOT). Mixed model ANOVAs were used to detect differences in SWAY mBESS and NeuroCom SOT scores with time (initial, one-week, final RTA) as the within-subjects factor and group (concussed, healthy) as the between-subjects factor. Spearman's Rho correlations explored the associations between NeuroCom SOT scores, SWAY scores, SCAT-5 symptom scores, and time in days to final RTA. Results: The sampled population was predominantly male and age (20 ± 1), and BMI differences were insignificant between groups. The SWAY did not detect differences between healthy and concussed participants and did not detect change over time [F(2,40) = .114, p = 0.89; F(2,40)= .276, p =0.60]. When assessing the relationship between the SWAY and the SOT, no correlation was found at any time point (r = -0.317 to -0.062, p > 0.05). Time to RTA demonstrated a moderate correlation with both SCAT-5 symptom severity score (r = .693, p < 0.01) and SCAT-5 total symptom score (r = .611, p < 0.01) at the one-week follow-up. Conclusion: The SWAY mBESS does not appear to be a valid balance assessment for the concussed patient. The SWAY mBESS in patients with concussion failed to demonstrate convergent validity and did not demonstrate an ability to validate known groups. When assessing the time to final RTA, the one-week post-initial assessment SCAT-5 symptom severity and total scores may help determine the length of recovery in this population. Level of Evidence: Level 3.
RESUMEN
As symbionts of animals, microbial eukaryotes benefit and harm their hosts in myriad ways. A model microeukaryote (Capsaspora owczarzaki) is a symbiont of Biomphalaria glabrata snails and may prevent transmission of parasitic schistosomes from snails to humans. However, it is unclear which host factors determine Capsaspora's ability to colonize snails. Here, we discovered that Capsaspora forms multicellular aggregates when exposed to snail hemolymph. We identified a molecular cue for aggregation: a hemolymph-derived phosphatidylcholine, which becomes elevated in schistosome-infected snails. Therefore, Capsaspora aggregation may be a response to the physiological state of its host, and it may determine its ability to colonize snails and exclude parasitic schistosomes. Furthermore, Capsaspora is an evolutionary model organism whose aggregation may be ancestral to animals. This discovery, that a prevalent lipid induces Capsaspora multicellularity, suggests that this aggregation phenotype may be ancient. Additionally, the specific lipid will be a useful tool for further aggregation studies.
RESUMEN
Adult germline stem cells are capable of self-renewal, tissue regeneration and production of large numbers of differentiated progeny. We show here that the classical mouse mutant luxoid affects adult germline stem cell self-renewal. Young homozygous luxoid mutant mice produce limited numbers of normal spermatozoa and then progressively lose their germ line after birth. Transplantation studies showed that germ cells from mutant mice did not colonize recipient testes, suggesting that the defect is intrinsic to the stem cells. We determined that the luxoid mutant contains a nonsense mutation in the gene encoding Plzf, a transcriptional repressor that regulates the epigenetic state of undifferentiated cells, and showed that Plzf is coexpressed with Oct4 in undifferentiated spermatogonia. This is the first gene shown to be required in germ cells for stem cell self-renewal in mammals.
Asunto(s)
Proteínas de Unión al ADN/genética , Espermatogonias/citología , Células Madre/citología , Factores de Transcripción/genética , Animales , Secuencia de Bases , Diferenciación Celular , Codón sin Sentido , ADN/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Factores de Transcripción de Tipo Kruppel , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Espermatogonias/metabolismo , Espermatogonias/trasplante , Células Madre/metabolismo , Testículo/citología , Factores de Transcripción/metabolismoRESUMEN
Background: Military physical therapists practicing direct-access routinely utilize diagnostic imaging and numerous published case reports demonstrate the ability of physical therapists to diagnose and appropriately disposition patients with foot/ankle and wrist/hand fractures. However, no larger cohort studies have explored the utilization of diagnostic imaging by physical therapists to detect fractures. Hypothesis/Purpose: To describe the utilization of diagnostic imaging in foot/ankle and wrist/hand injuries by physical therapists in a direct-access sports physical therapy clinic. Study Design: Retrospective cohort study. Methods: The Agfa Impax Client 6 image viewing software (IMPAX) was searched from 2014 to 2018 for patients with diagnostic imaging ordered for foot/ankle and wrist/hand injuries. The Armed Forces Health Longitudinal Technology Application (AHLTA) electronic medical record was independently reviewed by the principal and co-investigator physical therapists. Data extracted were demographics and elements from the patient history and physical examination. Results: In foot/ankle injuries, physical therapists diagnosed a fracture in 16% of the 177 cases and waited for an average of 3.9 days and 1.3 visits before ordering imaging. In wrist/hand injuries, physical therapists diagnosed a fracture in 24% of the 178 cases and waited for an average of 3.7 days and 1.2 visits before ordering imaging. The time to definitive care from the initial physical therapy evaluation was significantly different (p = 0.04) for foot/ankle fractures (0.6 days) compared to wrist/hand fractures (5.0 days). The Ottawa Ankle Rules demonstrated a negative likelihood ratio (-LR) of 0.11 (0.02, 0.72) and a positive likelihood ratio (+LR) of 1.99 (1.62, 2.44) for the diagnosis of foot/ankle fracture. Conclusions: Physical therapists utilizing diagnostic imaging in a direct-access sports physical therapy clinic diagnosed fractures in similar proportions for foot/ankle and wrist/hand injuries and quickly dispositioned patients to definitive care for those fractures. The diagnostic accuracy of the Ottawa Ankle Rules was similar to previously reported values. Level of Evidence: Level 3.
RESUMEN
Cardiovascular disease (CVD) is a leading cause of enhanced morbidity and mortality in persons with HIV (PWH) in the era of highly active antiretroviral therapy (AART). However, the underlying mechanisms are not fully understood. Regulatory T cells (Treg), notably the highly suppressive memory subset, have been shown to limit CVD. Importantly, memory Treg cell numbers remain low in many treated PWH. High density lipoproteins (HDL) also protect from CVD, and we previously found that Treg-HDL interactions reduce oxidative stress in these cells. Here, we evaluated Treg-HDL interactions in PWH and whether they were operative in those higher CVD risk. To do that, we recruited a cohort of PWH with intermediate/high CVD risk (median ASCVD risk score of 13.2%, n=15) or low/borderline risk (median ASCVD risk score of 3.6%, n=14), as well as a group of statins treated PWH with intermediate/high CVD risk (median ASCVD risk score of 12.7%, n=14). We evaluated Treg frequency, phenotype and response to HDL. PWH with Int/High CVD risk had a significantly lower number of memory Treg, but memory Treg were more activated and displayed an inflammatory phenotype, versus those with Low/BL CVD risk. In untreated patients, Treg absolute numbers were negatively correlated with ASCVD score. Although HDL decreased oxidative stress in memory Treg in all subjects, memory Treg from PWH with Int/High CVD risk were significantly less responsive to HDL than those from PWH with Low/BL CVD risk. The level of oxidative stress in memory Treg positively correlated with ASCVD scores. In contrast, plasma HDL from PWH, regardless of CVD risk, retained their anti-oxidative properties, suggesting that the defect in memory Treg response to HDL is intrinsic. Statin treatment partially ameliorated the memory Treg defect. In conclusion, the defective HDL-Treg interactions may contribute to the inflammation-induced increased CVD risk observed in many AART-treated PWH.
Asunto(s)
Enfermedades Cardiovasculares , Infecciones por VIH , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Humanos , Lipoproteínas HDL , Linfocitos T Reguladores , Enfermedades Cardiovasculares/complicaciones , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológicoRESUMEN
Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in cardiovascular disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies.
Asunto(s)
Apolipoproteína A-II/genética , Apolipoproteína A-II/aislamiento & purificación , Escherichia coli/metabolismo , Ingeniería Genética/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Apolipoproteína A-II/química , Apolipoproteína A-II/metabolismo , Dicroismo Circular , Técnicas de Cultivo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Expresión Génica , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Musculoskeletal injury (MSI) presents the greatest threat to military mission readiness. Atraumatic shoulder pain is a common military MSI that often results in persistent functional limitations. Shoulder orthopedic evaluation presents many diagnostic challenges, due in part to the possibility of a spinal source of symptoms. This case series outlines the use of mechanical diagnosis and therapy to screen the cervical and thoracic spine in active duty (AD) service members (SMs) with a chief complaint of unchanging or worsening shoulder pain. All three SMs previously received shoulder-specific diagnoses from experienced clinicians, yet repeated movements revealed a possible spinal nociceptive driver that guided targeted intervention. Treatment directed only at the cervical spine resulted in a clinically important improvement within an average of 10 days from the initial evaluation, return to duty (RTD) within an average of 32 days, and continued resolution at 3 months. SMs can independently complete the screening process with guidance from healthcare providers, ultimately shaping the treatment strategy and possibly facilitating self-management of future recurrence. This case series demonstrates that identification of shoulder pain of spinal source in the military population may be an important step in facilitating timely RTD. These cases also highlight the use of a standardized, systematic method to screen the cervical and thoracic spine that concurrently reveals the indicated treatment. Further research to determine the prevalence of shoulder pain of spinal source in the AD population and its impact on RTD rates has the potential to reduce the substantial burden of MSI in the military.