Sigma 1 receptor activation improves retinal structure and function in the RhoP23H/+ mouse model of autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of devastating inherited retinal diseases that leads to visual impairment and oftentimes complete blindness. Currently no cure exists for RP thus research into prolonging vision is imperative. Sigma 1 receptor (Sig1R) is a promising small molecule target that has neuroprotective benefits in retinas of rapidly-degenerating mouse models. It is not clear whether Sig1R activation can provide similar neuroprotective benefits in more slowly-progressing RP models. Here, we examined Sig1R-mediated effects in the slowly-progressing RhoP23H/+ mouse, a model of autosomal dominant RP. We characterized the retinal degeneration of the RhoP23H/+ mouse over a 10 month period using three in vivo methods: Optomotor Response (OMR), Electroretinogram (ERG), and Spectral Domain-Optical Coherence Tomography (SD-OCT). A slow retinal degeneration was observed in both male and female RhoP23H/+ mice when compared to wild type. The OMR, which reflects visual acuity, showed a gradual decline through 10 months. Interestingly, female mice had more reduction in visual acuity than males. ERG assessment showed a gradual decline in scotopic and photopic responses in RhoP23H/+ mice. To investigate the neuroprotective benefits of Sig1R activation in the RhoP23H/+ mouse model, mutant mice were treated with a high-specificity Sig1R ligand (+)-pentazocine ((+)-PTZ) 3x/week at 0.5 mg/kg and examined using OMR, ERG, SD-OCT. A significant retention of visual function was observed in males and females at 10 months of age, with treated females retaining ∼50% greater visual acuity than non-treated mutant females. ERG revealed significant retention of scotopic and photopic b-wave amplitudes at 6 months in male and female RhoP23H/+ mice treated with (+)-PTZ. Further, in vivo analysis by SD-OCT revealed a significant retention of outer nuclear layer (ONL) thickness in male and female treated RhoP23H/+ mice. Histological studies showed significant retention of IS/OS length (∼50%), ONL thickness, and number of rows of photoreceptor cell nuclei at 6 months in (+)-PTZ-treated mutant mice. Interestingly, electron microscopy revealed preservation of OS discs in (+)-PTZ treated mutant mice compared to non-treated. Taken collectively, the in vivo and in vitro data provide the first evidence that targeting Sig1R can rescue visual function and structure in the RhoP23H/+ mouse. These results are promising and provide a framework for future studies to investigate Sig1R as a potential therapeutic target in retinal degenerative disease.

ß-Arrestin pathway activation by selective ATR1 agonism promotes calcium influx in podocytes, leading to glomerular damage.

Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the ß-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated ß-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine ß-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated ß-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of ß-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the ß-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the ß-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated ß-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage.

Lipopolysaccharide Pretreatment Prevents Medullary Vascular Congestion following Renal Ischemia by Limiting Early Reperfusion of the Medullary Circulation.

BACKGROUND: Vascular congestion of the renal medulla-trapped red blood cells in the medullary microvasculature-is a hallmark finding at autopsy in patients with ischemic acute tubular necrosis. Despite this, the pathogenesis of vascular congestion is not well defined. METHODS: In this study, to investigate the pathogenesis of vascular congestion and its role in promoting renal injury, we assessed renal vascular congestion and tubular injury after ischemia reperfusion in rats pretreated with low-dose LPS or saline (control). We used laser Doppler flowmetry to determine whether pretreatment with low-dose LPS prevented vascular congestion by altering renal hemodynamics during reperfusion. RESULTS: We found that vascular congestion originated during the ischemic period in the renal venous circulation. In control animals, the return of blood flow was followed by the development of congestion in the capillary plexus of the outer medulla and severe tubular injury early in reperfusion. Laser Doppler flowmetry indicated that blood flow returned rapidly to the medulla, several minutes before recovery of full cortical perfusion. In contrast, LPS pretreatment prevented both the formation of medullary congestion and its associated tubular injury. Laser Doppler flowmetry in LPS-pretreated rats suggested that limiting early reperfusion of the medulla facilitated this protective effect, because it allowed cortical perfusion to recover and clear congestion from the large cortical veins, which also drain the medulla. CONCLUSIONS: Blockage of the renal venous vessels and a mismatch in the timing of cortical and medullary reperfusion results in congestion of the outer medulla's capillary plexus and promotes early tubular injury after renal ischemia. These findings indicate that hemodynamics during reperfusion contribute to the renal medulla's susceptibility to ischemic injury.

Transcriptome Analysis of Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Our previous studies have shown that systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spread to the eye with subsequent establishment of latency in choroid/RPE. In this study, RNA sequencing (RNA-Seq) analysis was used to determine the molecular genetic changes and pathways affected by ocular MCMV latency. MCMV (50 pfu per mouse) or medium as control were injected intra-peritoneally (i.p.) into BALB/c mice at <3 days after birth. At 18 months post injection, the mice were euthanized, and the eyes were collected and prepared for RNA-Seq. Compared to three uninfected control eyes, we identified 321 differentially expressed genes (DEGs) in six infected eyes. Using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA), we identified 17 affected canonical pathways, 10 of which function in neuroretinal signaling, with the majority of DEGs being downregulated, while 7 pathways function in upregulated immune/inflammatory responses. Retinal and epithelial cell death pathways involving both apoptosis and necroptosis were also activated. MCMV ocular latency is associated with upregulation of immune and inflammatory responses and downregulation of multiple neuroretinal signaling pathways. Cell death signaling pathways are also activated and contribute to the degeneration of photoreceptors, RPE, and choroidal capillaries.

MicroRNA cargo of extracellular vesicles released by skeletal muscle fibro-adipogenic progenitor cells is significantly altered with disuse atrophy and IL-1ß deficiency.

Fibro-adipogenic progenitor cells (FAPs) are a population of stem cells in skeletal muscle that play multiple roles in muscle repair and regeneration through their complex secretome; however, it is not well understood how the FAP secretome is altered with muscle disuse atrophy. Previous work suggests that the inflammatory cytokine IL-1ß is increased in FAPs with disuse and denervation. Inflammasome activation and IL-1ß secretion are also known to stimulate the release of extracellular vesicles (EVs). Here, we examined the microRNA (miRNA) cargo of FAP-derived, platelet-derived growth factor receptor A (PDGFRα+) EVs from hindlimb muscles of wild-type and IL-1ß KO mice after 14 days of single-hindlimb immobilization. Hindlimb muscles were isolated from mice following the immobilization period, and PDGFRα+ extracellular vesicles were isolated using size-exclusion chromatography and immunoprecipitation. Microarrays were performed to detect changes in miRNAs with unloading and IL-1ß deficiency. Results indicate that the PDGFRα+, FAP-derived EVs show a significant increase in miRNAs, such as miR-let-7c, miR-let-7b, miR-181a, and miR-124. These miRNAs have previously been demonstrated to play important roles in cellular senescence and muscle atrophy. Furthermore, the expression of these same miRNAs was not significantly altered in FAP-derived EVs isolated from the immobilized IL-1ß KO. These data suggest that disuse-related activation of IL-1ß can mediate the miRNA cargo of FAP-derived EVs, contributing directly to the release of senescence- and atrophy-related miRNAs. Therapies targeting FAPs in settings associated with muscle disuse atrophy may therefore have the potential to preserve muscle function and enhance muscle recovery.

Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Although pathologies associated with acute virus infections have been extensively studied, the effects of long-term latent virus infections are less well understood. Human cytomegalovirus, which infects 50% to 80% of humans, is usually acquired during early life and persists in a latent state for the lifetime. The purpose of this study was to determine whether systemic murine cytomegalovirus (MCMV) infection acquired early in life disseminates to and becomes latent in the eye and if ocular MCMV can trigger in situ inflammation and occurrence of ocular pathology. This study found that neonatal infection of BALB/c mice with MCMV resulted in dissemination of virus to the eye, where it localized principally to choroidal endothelia and pericytes and less frequently to the retinal pigment epithelium (RPE) cells. MCMV underwent ocular latency, which was associated with expression of multiple virus genes and from which MCMV could be reactivated by immunosuppression. Latent ocular infection was associated with significant up-regulation of several inflammatory/angiogenic factors. Retinal and choroidal pathologies developed in a progressive manner, with deposits appearing at both basal and apical aspects of the RPE, RPE/choroidal atrophy, photoreceptor degeneration, and neovascularization. The pathologies induced by long-term ocular MCMV latency share features of previously described human ocular diseases, such as age-related macular degeneration.

A Novel Mitochondrial Complex of Aldosterone Synthase, Steroidogenic Acute Regulatory Protein, and Tom22 Synthesizes Aldosterone in the Rat Heart.

Aldosterone, which regulates renal salt retention, is synthesized in adrenocortical mitochondria in response to angiotensin II. Excess aldosterone causes myocardial injury and heart failure, but potential intracardiac aldosterone synthesis has been controversial. We hypothesized that the stressed heart might produce aldosterone. We used blue native gel electrophoresis, immunoblotting, protein crosslinking, coimmunoprecipitations, and mass spectrometry to assess rat cardiac aldosterone synthesis. Chronic infusion of angiotensin II increased circulating corticosterone levels 350-fold and induced cardiac fibrosis. Angiotensin II doubled and telmisartan inhibited aldosterone synthesis by heart mitochondria and cardiac production of aldosterone synthase (P450c11AS). Heart aldosterone synthesis required P450c11AS, Tom22 (a mitochondrial translocase receptor), and the intramitochondrial form of the steroidogenic acute regulatory protein (StAR); protein crosslinking and coimmunoprecipitation studies showed that these three proteins form a 110-kDa complex. In steroidogenic cells, extramitochondrial (37-kDa) StAR promotes cholesterol movement from the outer to inner mitochondrial membrane where cholesterol side-chain cleavage enzyme (P450scc) converts cholesterol to pregnenolone, thus initiating steroidogenesis, but no function has previously been ascribed to intramitochondrial (30-kDa) StAR; our data indicate that intramitochondrial 30-kDa StAR is required for aldosterone synthesis in the heart, forming a trimolecular complex with Tom22 and P450c11AS. This is the first activity ascribed to intramitochondrial StAR, but how this promotes P450c11AS activity is unclear. The stressed heart did not express P450scc, suggesting that circulating corticosterone (rather than intracellular cholesterol) is the substrate for cardiac aldosterone synthesis. Thus, the stressed heart produced aldosterone using a previously undescribed intramitochondrial mechanism that involves P450c11AS, Tom22, and 30-kDa StAR. SIGNIFICANCE STATEMENT: Prior studies of potential cardiac aldosterone synthesis have been inconsistent. This study shows that the stressed rat heart produces aldosterone by a novel mechanism involving aldosterone synthase, Tom22, and intramitochondrial steroidogenic acute regulatory protein (StAR) apparently using circulating corticosterone as substrate. This study establishes that the stressed rat heart produces aldosterone and for the first time identifies a biological role for intramitochondrial 30-kDa StAR.

Ocular cytomegalovirus latency exacerbates the development of choroidal neovascularization.

Age-related macular degeneration (AMD) is a complex, multifactorial, progressive disease which represents a leading cause of irreversible visual impairment and blindness in older individuals. Human cytomegalovirus (HCMV), which infects 50-80% of humans, is usually acquired during early life and persists in a latent state for the life of the individual. In view of its previously described pro-angiogenic properties, we hypothesized that cytomegalovirus might be a novel risk factor for progression to an advanced form, neovascular AMD, which is characterized by choroidal neovascularization (CNV). The purpose of this study was to investigate if latent ocular murine cytomegalovirus (MCMV) infection exacerbated the development of CNV in vascular endothelial growth factor (VEGF)-overexpressing VEGF-Ahyper mice. Here we show that neonatal infection with MCMV resulted in dissemination of virus to various organs throughout the body including the eye, where it localized principally to the choroid in both VEGF-overexpressingVEGF-Ahyper and wild-type(WT) 129 mice. By 6 months post-infection, no replicating virus was detected in eyes and extraocular tissues, although virus DNA was still present in all eyes and extraocular tissues of both VEGF-Ahyper and WT mice. Expression of MCMV immediate early (IE) 1 mRNA was detected only in latently infected eyes of VEGF-Ahyper mice, but not in eyes of WT mice. Significantly increased CNV was observed in eyes of MCMV-infected VEGF-Ahyper mice compared to eyes of uninfected VEGF-Ahyper mice, while no CNV lesions were observed in eyes of either infected or uninfected WT mice. Protein levels of several inflammatory/angiogenic factors, particularly VEGF and IL-6, were significantly higher in eyes of MCMV-infected VEGF-Ahyper mice, compared to uninfected controls. Initial studies of ocular tissue from human cadavers revealed that HCMV DNA was present in four choroid/retinal pigment epithelium samples from 24 cadavers. Taken together, our data suggest that ocular HCMV latency could be a significant risk factor for the development of AMD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Role of Caspase-12 in Retinal Bystander Cell Death and Innate Immune Responses against MCMV Retinitis.

(1) Background: caspase-12 is activated during cytomegalovirus retinitis, although its role is presently unclear. (2) Methods: caspase-12-/- (KO) or caspase-12+/+ (WT) mice were immunosup eyes were analyzed by plaque assay, TUNEL assay, immunohistochemical staining, western blotting, and real-time PCR. (3) Results: increased retinitis and a more extensive virus spread were detected in the retina of infected eyes of KO mice compared to WT mice at day 14 p.i. Compared to MCMV injected WT eyes, mRNA levels of interferons α, ß and γ were significantly reduced in the neural retina of MCMV-infected KO eyes at day 14 p.i. Although similar numbers of MCMV infected cells, similar virus titers and similar numbers of TUNEL-staining cells were detected in injected eyes of both KO and WT mice at days 7 and 10 p.i., significantly lower amounts of cleaved caspase-3 and p53 protein were detected in infected eyes of KO mice at both time points. (4) Conclusions: caspase-12 contributes to caspase-3-dependent and independent retinal bystander cell death during MCMV retinitis and may also play an important role in innate immunity against virus infection of the retina.

Oral NaHCO3 Activates a Splenic Anti-Inflammatory Pathway: Evidence That Cholinergic Signals Are Transmitted via Mesothelial Cells.

We tested the hypothesis that oral NaHCO3 intake stimulates splenic anti-inflammatory pathways. Following oral NaHCO3 loading, macrophage polarization was shifted from predominantly M1 (inflammatory) to M2 (regulatory) phenotypes, and FOXP3+CD4+ T-lymphocytes increased in the spleen, blood, and kidneys of rats. Similar anti-inflammatory changes in macrophage polarization were observed in the blood of human subjects following NaHCO3 ingestion. Surprisingly, we found that gentle manipulation to visualize the spleen at midline during surgical laparotomy (sham splenectomy) was sufficient to abolish the response in rats and resulted in hypertrophy/hyperplasia of the capsular mesothelial cells. Thin collagenous connections lined by mesothelial cells were found to connect to the capsular mesothelium. Mesothelial cells in these connections stained positive for the pan-neuronal marker PGP9.5 and acetylcholine esterase and contained many ultrastructural elements, which visually resembled neuronal structures. Both disruption of the fragile mesothelial connections or transection of the vagal nerves resulted in the loss of capsular mesothelial acetylcholine esterase staining and reduced splenic mass. Our data indicate that oral NaHCO3 activates a splenic anti-inflammatory pathway and provides evidence that the signals that mediate this response are transmitted to the spleen via a novel neuronal-like function of mesothelial cells.

Inflammation and outer blood-retina barrier (BRB) compromise following choroidal murine cytomegalovirus (MCMV) infections.

Purpose: The purpose of this study was to determine whether the blood-retina barrier is compromised by choroidal murine cytomegalovirus (MCMV) infection, using electron microscopy. Methods: BALB/c mice were immunosuppressed with methylprednisolone and monoclonal antibodies to CD4 and CD8. At several time points post-MCMV intraperitoneal inoculation, the eyes were removed and analyzed with western blotting and immunoelectron microscopy for the presence of MCMV early antigen (EA) and the host protein RIP3. Posterior eyecups from RIP3-/- and RIP3+/+ mice were cultured and inoculated with MCMV. At days 4, 7, and 11 post-infection, cultures were collected and analyzed with plaque assay, immunohistochemical staining, and real-time PCR (RT-PCR). Results: MCMV EA was observed in the nuclei of vascular endothelial cells and pericytes in the choriocapillaris. Disruption of Bruch's membrane was observed, especially at sites adjacent to activated platelets, and a few RPE cells containing some enlarged vesicles were found directly beneath disrupted Bruch's membrane. Some virus particles were also observed in the enlarged vesicles of RPE cells. Levels of the RIP3 protein, which was observed mainly in the RPE cells and the basement membrane of the choriocapillaris, were greatly increased following MCMV infection, while depletion of RIP3 resulted in greatly decreased inflammasome formation, as well as expression of downstream inflammation factors. Conclusions: The results suggest that systemic MCMV spreads to the choroid and replicates in vascular endothelia and pericytes of the choriocapillaris during immunosuppression. Choroidal MCMV infection is associated with in situ inflammation and subsequent disruption of Bruch's membrane and the outer blood-retina barrier.

Innate lymphoid cells: a paradigm for low SSI in cleft lip repair.

BACKGROUND: Cleft lip and palate reconstructions demonstrate significantly lower surgical site infection rates compared with clean-contaminated cases, prompting investigation into the pathophysiology causing this discrepancy. Recent studies have identified a new group of innate lymphocytes called innate lymphoid cells (ILCs), located in barrier surfaces of the skin, airways, and intestine. Our objectives were to explore for the first time the presence of ILCs in the vermillion of neonates and young children undergoing cleft lip reconstruction and characterize their composition by measuring the three classes of ILCs. MATERIALS AND METHODS: Lip tissue samples were collected from 13 subjects undergoing vermillion resection during cleft lip reconstructive surgery. Preparative, transmission electron microscopy, and analytical flow cytometry were performed. The functionality of ILCs was tested in terms of their capacity to produce type 1 (IFN-γ/TNF-α), type 2 (IL-5/IL-13), and type 3 (IL-17/IL-22) cytokines. Data were analyzed using Student t test or the analysis of variance to establish significance (P < 0.05) among groups for all other data. RESULTS: All three classes of ILCs were detected and visualized in the tissue samples. In all samples, the level of ILC2 subset was significantly higher than the other two ILC subsets (P < 0.01), followed by the ILC1 subset, which was present in significantly higher levels than the ILC3 subset (P < 0.05). CONCLUSIONS: Our data place ILCs for the first time in the interface of oral mucosal immunity, tissue microenvironment, and homeostasis during and after tissue development, possibly explaining lower infection rates in cleft lip or palate reconstructions.

The Number of Trials Required to Obtain a Representative Movement Pattern During a Hurdle Hop Exercise.

When reporting a subject's mean movement pattern, it is important to ensure that reported values are representative of the subject's typical movement. While previous studies have used the mean of 3 trials, scientific justification of this number is lacking. One approach is to determine statistically how many trials are required to achieve a representative mean. This study compared 4 methods of calculating the number of trials required in a hopping movement to achieve a representative mean. Fifteen males completed 15 trials of a lateral hurdle hop. Range of motion at the trunk, pelvis, hip, knee, and ankle, in addition to peak moments for the latter 3 joints were examined. The number of trials required was computed using a peak intraclass correlation coefficient method, sequential analysis with a bandwidth of acceptable variance in the mean, and a novel method based on the standard error of measurement (SEMind). The number of trials required across all variables ranged from 2 to 12 depending on method, joint, and anatomical plane. The authors advocate the SEMind method as it demonstrated fewer limitations than the other methods. Using the SEMind, the required number of trials for a representative mean during the lateral hurdle hop is 6.

Can a Single-Leg Squat Provide Insight Into Movement Control and Loading During Dynamic Sporting Actions in Patients With Athletic Groin Pain?

CONTEXT: Chronic athletic groin pain (AGP) is common in field sports and has been associated with abnormal movement control and loading of the hip and pelvis during play. A single-leg squat (SLS) is commonly used by clinicians to assess movement control, but whether it can provide insight into control during more dynamic sporting movements in AGP patients is unclear. OBJECTIVE: To determine the relationships between biomechanical measures in an SLS and the same measures in a single-leg drop landing, single-leg hurdle hop, and a cutting maneuver in AGP patients. DESIGN: Cross-sectional study. SETTING: Biomechanics laboratory. PATIENTS: 40 recreational field-sports players diagnosed with AGP. INTERVENTION: A biomechanical analysis of each individual's SLS, drop landing, hurdle hop, and cut was undertaken. MAIN OUTCOME MEASURES: Hip, knee, and pelvis angular displacement and hip and knee peak moments. Pearson product-moment correlations were used to examine relationships between SLS measures and equivalent measures in the other movements. RESULTS: There were no significant correlations between any hip or pelvis measure in the SLS with the same measures in the drop landing, hurdle hop, or cut (r = .03-.43, P > .05). Knee frontal- and transverse-plane angular displacement were related in the SLS and drop landing only, while knee moments were related in the SLS, drop-landing, and hurdle hop (r = .50-.67, P < .05). CONCLUSION: For AGP patients, an SLS did not provide meaningful insight into hip and pelvis control or loading during sporting movements that are associated with injury development. The usefulness of an SLS test in the assessment of movement control and loading in AGP patients is thus limited. The SLS provided moderate insight into knee control while landing and therefore may be of use in the examination of knee-injury risk.

Isokinetic muscle strength and readiness to return to sport following anterior cruciate ligament reconstruction: is there an association? A systematic review and a protocol recommendation.

INTRODUCTION: Following anterior cruciate ligament reconstruction (ACLR), strength is a key variable in regaining full function of the knee. Isokinetic strength is commonly used as part of the return to sport (RTS) criteria. AIM: We systematically reviewed the isokinetic strength evaluation protocols that are currently being used following ACLR. A secondary aim was to suggest an isokinetic protocol that could meet RTS criteria. METHOD: Articles were searched using ScienceDirect, PubMed and Sage Journals Online, combined with cross-checked reference lists of the publications. Protocol data and outcome measurements and RTS criteria were extracted from each article included in the review. RESULTS: 39 studies met the inclusion criteria and reported their isokinetic strength evaluation protocol following ACLR. The variables that were most commonly used were concentric/concentric mode of contraction (31 studies), angular velocity of 60°/s (29 studies), 3-5 repetitions (24 studies), range of motion of 0-90° (6 studies), and using gravity correction (9 studies). 8 studies reported strength limb symmetry index scores as part of their RTS criteria. CONCLUSIONS: There was no standardised isokinetic protocol following ACLR; isokinetic strength measures have not been validated as useful predictors of successful RTS. We propose a standard protocol to allow consistency of testing and accurate comparison of future research.

Biomechanical Factors Associated With Jump Height: A Comparison of Cross-Sectional and Pre-to-Posttraining Change Findings.

Previous studies investigating the biomechanical factors associated with maximal countermovement jump height have typically used cross-sectional data. An alternative but less common approach is to use pre-to-posttraining change data, where the relationship between an improvement in jump height and a change in a factor is examined more directly. Our study compared the findings of these approaches. Such an evaluation is necessary because cross-sectional studies are currently a primary source of information for coaches when examining what factors to train to enhance performance. The countermovement jump of 44 males was analyzed before and after an 8-week training intervention. Correlations with jump height were calculated using both cross-sectional (pretraining data only) and pre-to-posttraining change data. Eight factors identified in the cross-sectional analysis were not significantly correlated with a change in jump height in the pre-to-post analysis. Additionally, only 6 of 11 factors identified in the pre-to-post analysis were identified in the cross-sectional analysis. These findings imply that (a) not all factors identified in a cross-sectional analysis may be critical to jump height improvement and (b) cross-sectional analyses alone may not provide an insight into all of the potential factors to train to enhance jump height. Coaches must be aware of these limitations when examining cross-sectional studies to identify factors to train to enhance jump ability. Additional findings highlight that although exercises prescribed to improve jump height should aim to enhance concentric power production at all joints, a particular emphasis on enhancing hip joint peak power may be warranted.

Interplay of autophagy and apoptosis during murine cytomegalovirus infection of RPE cells.

PURPOSE: Previous studies have demonstrated that autophagy is involved in the pathogenesis of human cytomegalovirus (HCMV) infection. However, whether autophagy is regulated by murine cytomegalovirus (MCMV) infection has not yet been investigated. The purpose of these studies was to determine how autophagy is affected by MCMV infection of the retinal pigment epithelial (RPE) cells and whether there is a functional relationship between autophagy and apoptosis; and if so, how regulation of autophagy impacts apoptosis. METHODS: RPE cells were isolated from C57BL/6 mice and infected with MCMV K181. The cells were cultured in medium containing rapamycin, chloroquine, or ammonium chloride. Green fluorescent protein-light chain 3 (GFP-LC3) plasmid was transfected to RPE cells, and the GFP-LC3 positive puncta were counted. Electron microscopic (EM) images were taken to visualize the structure of the autophagic vacuoles. Western blot was performed to detect the expression of related proteins. Trypan blue exclusion assay was used to measure the percentage of viable cells. RESULTS: Although the LC3B-II levels consistently increased during MCMV infection of RPE cells, administration of chloroquine or ammonium chloride increased LC3B-II expression only at the early stage of infection (6 h post-inoculation [p.i.] and 12 h p.i.), not at or after 24 h p.i. The punctate autophagic vacuoles in the GFP-LC3 transfected RPE cells were counted using light microscopy or by EM examination, the number of autophagic vacuoles was significantly increased in the MCMV-infected RPE cells compared to the uninfected controls. Compared to untreated MCMV-infected control cells, rapamycin treatment resulted in a significant decrease in the cleaved caspase 3 levels as well as a significant decrease in the ratio of phosphorylated mammalian target of rapamycin (mTOR) to total mTOR and in the ratio of phosphorylated P70S6K to total P70S6K. In contrast, chloroquine treatment resulted in a significant increase in the cleaved caspase 3 levels in the MCMV-infected RPE cells. CONCLUSIONS: Autophagic vacuole accumulation was detected during MCMV infection of RPE cells. In contrast, autophagic flux was greatly decreased at or after 24 h p.i. The results suggest that MCMV might have a strategy for inhibiting or blocking autophagy activity by targeting a later autophagy process, such as the formation of autolysosomes or degradation of their content. Our data also suggest that there is a functional relationship between autophagy and apoptosis, which plays an important role during MCMV infection of the RPE.

Biomechanical factors associated with time to complete a change of direction cutting maneuver.

Cutting ability is an important aspect of many team sports, however, the biomechanical determinants of cutting performance are not well understood. This study aimed to address this issue by identifying the kinetic and kinematic factors correlated with the time to complete a cutting maneuver. In addition, an analysis of the test-retest reliability of all biomechanical measures was performed. Fifteen (n = 15) elite multidirectional sports players (Gaelic hurling) were recruited, and a 3-dimensional motion capture analysis of a 75° cut was undertaken. The factors associated with cutting time were determined using bivariate Pearson's correlations. Intraclass correlation coefficients (ICCs) were used to examine the test-retest reliability of biomechanical measures. Five biomechanical factors were associated with cutting time (2.28 ± 0.11 seconds): peak ankle power (r = 0.77), peak ankle plantar flexor moment (r = 0.65), range of pelvis lateral tilt (r = -0.54), maximum thorax lateral rotation angle (r = 0.51), and total ground contact time (r = -0.48). Intraclass correlation coefficient scores for these 5 factors, and indeed for the majority of the other biomechanical measures, ranged from good to excellent (ICC >0.60). Explosive force production about the ankle, pelvic control during single-limb support, and torso rotation toward the desired direction of travel were all key factors associated with cutting time. These findings should assist in the development of more effective training programs aimed at improving similar cutting performances. In addition, test-retest reliability scores were generally strong, therefore, motion capture techniques seem well placed to further investigate the determinants of cutting ability.

Analysis of characterizing phases on waveform: an application to vertical jumps.

The aim of this study is to propose a novel data analysis approach, an analysis of characterizing phases (ACP), that detects and examines phases of variance within a sample of curves utilizing the time, magnitude, and magnitude-time domains; and to compare the findings of ACP to discrete point analysis in identifying performance-related factors in vertical jumps. Twenty-five vertical jumps were analyzed. Discrete point analysis identified the initial-to-maximum rate of force development (P=.006) and the time from initial-to-maximum force (P=.047) as performance-related factors. However, due to intersubject variability in the shape of the force curves (ie, non-, uni- and bimodal nature), these variables were judged to be functionally erroneous. In contrast, ACP identified the ability to apply forces for longer (P<.038), generate higher forces (P<.027), and produce a greater rate of force development (P<.003) as performance-related factors. Analysis of characterizing phases showed advantages over discrete point analysis in identifying performance-related factors because it (i) analyses only related phases, (ii) analyses the whole data set, (iii) can identify performance-related factors that occur solely as a phase, (iv) identifies the specific phase over which differences occur, and (v) analyses the time, magnitude and combined magnitude-time domains.

Targeting Chemoresistance in Advanced Bladder Cancers with a Novel Adjuvant Strategy.

Advanced urinary bladder cancer is characterized by rapid progression and development of therapy resistance. About 30% of the patients are diagnosed with high-grade tumors (grade > T2a). A typical nonsurgical treatment is systemic chemotherapy using cisplatin (C) and gemcitabine (G). However, treatment failure and subsequent disease progression are common in treated patients, and adjuvant therapies are not significantly effective. The therapeutic potential of a molecular hybrid of ursolic acid (UA), a pentacyclic-triterpene conjugated to N-methyl piperazine (UA4), was tested on both naïve (WT) and gemcitabine-resistant (GemR) variants of two human invasive bladder cancer cell lines, 5637 and T24. UA4 killed 5637 (4 µmol/L), T24 (4 µmol/L) WT, and GemR cells in vitro at equal potency. Pretreatment with UA4 followed by G synergistically killed WT and GemR cells by >50% compared with G followed by UA4. Oral gavage of UA4 (100 mg/kg) inhibited WT and GemR tumor growth in athymic mice. UA4 + G was more effective against GemR tumors than either drug alone. Studies revealed cytotoxic autophagy as a mechanism of UA4 cytotoxicity. UA4 induced moderate apoptosis in T24 but not in 5637 cells. Mitochondrial integrity and function were most affected by UA4 because of high levels of reactive oxygen species, disruption of mitochondrial membrane, and cell cycle arrest. These effects were enhanced in the UA4 + G combination. UA4 was well-tolerated in mice, and oral gavage led to a serum level >1 µmol/L with no systemic toxicity. These results show the potential of UA4 as a nontoxic alternative treatment for high-grade bladder cancer.