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Background Humeral retrotorsion (HRT) is one bony adaptation that occurs in overhead athletes. This bony adaptation often leads to bilateral changes in range of motion at the glenohumeral joint. Because HRT can create different stress environments on the surrounding tissue, it may play a role in upper-limb injury and pain profiles. Therefore, the aim of this review was to examine whether HRT plays a role in shoulder and elbow injury profiles. Methods Two separate critical appraisal tools were administered: the Newcastle-Ottawa Scale (case control) and the Appraisal Tool for Cross-sectional Studies. The primary author extracted all data and obtained means and standard deviations for each outcome. Cohen d effect sizes (ESs) were calculated (ES [95% confidence interval]) for all HRT measurements including nondominant, dominant, and side-to-side differences. Finally, the Strength of Recommendation Taxonomy was used to evaluate the overall strength of the recommendation. Results Nine articles were included in this review. Large ESs were present in 2 studies on examination of symptomatic and asymptomatic dominant HRT and ranged between 0.83 (0.08-1.55) and –2.57 (–3.66 to 1.99). The majority of all ESs for all HRT measurements were moderate or low, rendering comparisons between asymptomatic and symptomatic cohorts that were not clinically meaningful. Conclusion The Strength of Recommendation Taxonomy rating was C based on inconsistent findings. Differences in sports populations and definitions of injuries across studies may be one reason for the varying ESs. HRT does occur in the overhead population, but the degree to which this HRT starts to affect upper-limb injury is unknown and is more than likely player specific and multifactorial.

Ocular tissues possess a robust antioxidant defence system to minimize oxidative stress and preserve tissue structure and function. Glutathione (GSH) is a powerful antioxidant and in the lens exists at unusually high concentrations. However, with advancing age, GSH levels deplete specifically in the lens centre initiating a chain of biochemical events that ultimately result in protein aggregation, light scattering and age-related nuclear cataract. However, antioxidant supplementation has been shown to be ineffective in preventing or delaying cataract indicating that a better understanding of the delivery, uptake and metabolism of GSH in the different regions of the lens is required. This information is essential for the development of scientifically informed approaches that target the delivery of GSH to the lens nucleus, the region of the lens most affected by age-related cataract.

BACKGROUND Cystine/glutamate exchanger (xCT) knockout mice are reported to exhibit an oxidative shift in the plasma cystine/cysteine ratio reminiscent of that seen in human plasma of ageing individuals. This suggests that the xCT knockout mouse is a model of accelerated ageing. The aim of this study was to examine the progression of age-related pathologies in the ocular tissues of wild-type mice and compare this to the xCT knockout mice. METHODS Wild-type and xCT knockout mice were examined longitudinally or as separate groups of animals at six weeks, three months, six months, nine months, and 12 months of age. All groups of mice were anaesthetised, intraocular pressure measured using the iCare TONOLAB rebound tonometer and eyes examined using the Micron IV system. RESULTS While the aim of the study was to determine if xCT knockout mice developed age-related pathologies earlier than wild-type mice, it was inadvertently discovered in the longitudinal cohort of animals, that the eyes developed corneal lesions in both groups of animals by six months of age, which obscured examination of the lens and retina. These lesions were not characteristic of age-related pathologies, but rather due to an external stressor. Lesions in the xCT knockout mice developed at an earlier age compared to wild-type mice, suggesting that loss of xCT exacerbates damage to the cornea, most likely caused by the rebound tonometer. When the same ocular procedures were performed on separate cohorts of mice of specific ages, no corneal lesions were detected for both groups of mice. CONCLUSIONS While it may seem advantageous to examine the same cohort of mice to monitor the development of age-related pathologies, the type of ophthalmic tests conducted needs to be carefully considered to avoid introducing pathologies that are inadvertently a result of the examination process itself.

Freiberg’s disease is a rare condition characterized by avascular necrosis of the lesser metatarsal heads. The incidence is highest in females, and the disease is usually unilateral, affecting just a single metatarsal. In this report, we describe the first male case of Freiberg’s disease involving the second and third metatarsals in both feet, with the aim of highlighting the condition as a potential cause of such forefoot pain. One year of follow-up is provided.

Age related nuclear (ARN) cataracts in humans take years to form and so experimental models have been developed to mimic the process in animals as a means of better understanding the etiology of nuclear cataracts in humans. A major limitation with these animal models is that many of the biochemical and physiological changes are not typical of that seen in human ARN cataract. In this review, we highlight the work of Frank Giblin and colleagues who established an in vivo animal model that replicates many of the changes observed in human ARN cataract. This model involves exposing aged guinea pigs to hyperbaric oxygen (HBO), which by causing the depletion of the antioxidant glutathione (GSH) specifically in the lens nucleus, produces oxidative changes to nuclear proteins, nuclear light scattering and a myopic shift in lens power that mimics the change that often precedes cataract development in humans. However, this model involves multiple HBO treatments per week, with sometimes up to a total of 100 treatments, spanning up to eight months, which is both costly and time consuming. To address these issues, Giblin developed an in vitro model that used rabbit lenses exposed to HBO for several hours which was subsequently shown to replicate many of the changes observed in human ARN cataract. These experiments suggest that HBO treatment of in vitro animal lenses may serve as a more economical and efficient model to study the development of cataract. Inspired by these experiments, we investigated whether exposure of young bovine lenses to HBO for 15 h could also serve as a suitable acute model of ARN cataract. We found that while this model is able to exhibit some of the biochemical and physiological changes associated with ARN cataract, the decrease in lens power we observed was more characteristic of the hyperopic shift in refraction associated with ageing. Future work will investigate whether HBO treatment to age the bovine lens in combination with an oxidative stressor such as UV light will induce refractive changes more closely associated with human ARN cataract. This will be important as developing an animal model that replicates the changes to lens biochemistry, physiology and optics observed in human ARN cataracts is urgently required to facilitate the identification and testing of anti-cataract therapies that are effective in humans.

Purpose To use magnetic resonance imaging (MRI) to measure age-dependent changes in total and free water in human lenses in vivo. Methods Sixty-four healthy adults aged 18 to 86 years were recruited, fitted with a 32-channel head receiver coil, and placed in a 3 Tesla clinical MR scanner. Scans of the crystalline lens were obtained using a volumetric interpolated breath-hold examination sequence with dual flip angles, which were corrected for field inhomogeneity post-acquisition using a B1-map obtained using a turbo-FLASH sequence. The spatial distribution and content of corrected total (ρlens) and free (T1) water along the lens optical axis were extracted using custom-written code. Results Lens total water distribution and content did not change with age (all P > 0.05). In contrast to total water, a gradient in free water content that was highest in the periphery relative to the center was present in lenses across all ages. However, this initially parabolic free water gradient gradually developed an enhanced central plateau, as indicated by increasing profile shape parameter values (anterior: 0.067/y, P = 0.004; posterior: 0.050/y, P = 0.020) and central free water content (1.932 ms/y, P = 0.022) with age. Conclusions MRI can obtain repeatable total and free water measurements of in vivo human lenses. The observation that the lens steady-state free, but not total, water gradient is abolished with age raises the possibility that alterations in protein-water interactions are an underlying cause of the degradation in lens optics and overall vision observed with aging.

Purpose The purpose of this study was to determine the importance of the xCT is a subunit. The cystine/glutamate antiporter is actually system xc-xCT subunit of the cystine/glutamate antiporter in maintaining redox balance by investigating the effects of the loss of xCT on lens transparency and cystine/cysteine balance in the aqueous humour. Methods C57Bl/6 wild-type and xCT knockout mice at five age groups (6 weeks to 12 months) were used. Lens transparency was examined using a slit-lamp and morphological changes visualized by immunolabelling and confocal microscopy. Quantification of glutathione in lenses and cysteine and cystine levels in the aqueous was conducted by liquid chromatography tandem mass spectrometry (LC-MS/MS). Results Slit-lamp examinations revealed that 3-month-old wild-type mice and xCT knockout mice lenses exhibited an anterior localized cataract. The frequency of this cataract significantly increased in the knockout mice compared to the wild-type mice. Morphological studies revealed a localized swelling of the lens fiber cells at the anterior pole. Glutathione levels in whole lenses were similar between wild-type and knockout mice. However, glutathione levels were significantly decreased at 3 months in the knockout mice in the lens epithelium compared to the wild-type mice. Aqueous cysteine levels remained similar between wild-type and knockout mice at all age groups, whereas cystine levels were significantly increased in 3-, 9-, and 12-month-old knockout mice compared to wild-type mice. Conclusions Loss of xCT resulted in the depletion of glutathione in the epithelium and an oxidative shift in the cysteine/cystine ratio of the aqueous. Together, these oxidative changes may contribute to the accelerated development of an anterior cataract in knockout mice, which appears to be a normal feature of aging in wild-type mice.

Study Design Clinical case series. Objective To assess objective outcomes of surgical correction of post–external beam radiation therapy (ERBT) kyphosis in a series of five adults. Summary of Background Data EBRT is a well-established treatment for many cancers in children and adults. One complication associated with EBRT is postirradiation spine deformity. Scoliosis is the most common deformity, but kyphosis also occurs frequently. Differences in deformity patterns are likely related to the location and intensity of radiation. To our knowledge, no studies have addressed treatment of these deformities in adults, and the most recent case series (of children) was published in 2005. Methods We present a series of five adults who underwent surgery for postirradiation kyphosis, with a mean follow-up of 3.8 years (range, 2.5–6.2 years). Results Surgery improved the kyphotic deformity in all patients. Overall mean kyphotic deformity correction was 56° and was larger for cervical/cervicothoracic deformities (mean, 76°) than for lumbar deformities (mean, 42°) at midterm follow-up. Patients reported significant improvements in pain and self-image. Consistent with prior case series of children, we observed a high rate of complications (mean, 1.4 complications per patient) in adults. Three patients each underwent an unplanned surgical procedure because of a complication. Conclusion The surgical treatment of postirradiation kyphotic spinal deformity is challenging, with common postoperative complications such as infection, instrumentation failure, and pseudarthrosis. However, with modern surgical techniques and spinal instrumentation, excellent deformity correction can be achieved and maintained. We recommend performing a two-stage procedure for cervicothoracic deformity, with anterior release followed by posterior fusion and instrumentation. In thoracolumbar deformities, correction can be achieved through single-stage posterior fusion. Rigid spinopelvic fixation with sacral-alar-iliac screws and second-stage anterior lumbar interbody fusion at L5–S1 is recommended to reduce nonunion risk. Cement augmentation of proximal and distal anchors can help prevent junctional failure. Level of Evidence Level IV.

Schwannomatosis is a multiple tumor syndrome in which patients develop benign tumors along peripheral nerves throughout the body. The first symptom with which schwannomatosis patients often present, prior to discovery of tumors, is pain. This pain can be debilitating and is often inadequately alleviated by pharmacological approaches. Schwannomatosis-associated pain can be localized to the area of a tumor, or widespread. Moreover, not all tumors are painful, and the occurrence of pain is often unrelated to tumor size or location. We speculate that some individual tumors, but not others, secrete factors that act on nearby nerves to augment nociception by producing neuronal sensitization or spontaneous neuronal firing. We created cell lines from human SWN tumors with varying degrees of pain. We have found that conditioned medium (CM) collected from painful SWN tumors, but not that from nonpainful SWN tumors, sensitized DRG neurons, causing increased sensitivity to depolarization by KCl, increased response to noxious TRPV1 and TRPA1 agonists and also upregulated the expression of pain-associated genes in DRG cultures. Multiple cytokines were also detected at higher levels in CM from painful tumors. Taken together our data demonstrate a differential ability of painful versus non-painful human schwannomatosis tumor cells to secrete factors that augment sensory neuron responsiveness, and thus identify a potential determinant of pain heterogeneity in schwannomatosis.

The cystine-glutamate exchanger (system xc-) is responsible for the exchange of extracellular cystine for intracellular glutamate. In this study, we mapped the expression of xCT, the light chain subunit of system xc- in the different tissues of 3-6-week-old mouse (C57BL/6J) eye and have used an xCT knockout mouse to verify labelling specificity. Moreover, using the xCT knockout mouse, we investigated whether xCT was involved in maintaining extracellular redox balance in the eye. xCT transcript and protein were present in the cornea, lens and retina of wild-type mice, but not knockout mice. xCT was localised to the corneal epithelium, and the lens epithelium and cortical fibre cells but was absent in the iris. xCT localisation could not be determined in the ciliary body or retina, since xCT labelling was also detected in the knockout indicating a lack of specificity of the xCT antibody in tissues of a neural origin. Intracellular cysteine and cystine concentrations were similar in the wild-type and xCT knockout mouse for the cornea, lens, and retina. While extracellular cysteine levels were similar between the plasma, aqueous humour, and vitreous humour of the wild-type and xCT knockout mouse, extracellular cystine levels in the plasma and aqueous were significantly elevated in the xCT knockout mouse relative to the wild type. This suggests that loss of xCT results in an increased oxidative environment, particularly within the anterior chamber of the eye in which the aqueous humour resides. How this oxidative shift impacts ocular tissues that interface with the aqueous humour over time will be the focus of future work.

Changes to cellular metabolism often precede changes to other biomolecules that in turn cause tissue pathologies. In the ocular lens, changes in the spatial distribution and concentration of metabolites that are associated with the aging process are implicated in the development of the primary lens pathology, lens cataract. The development of age-related nuclear cataract is characterised by regional changes to protein cross-linking and protein insolubilisation that subsequently results in cataract formation. In the present study, MALDI FT-ICR imaging mass spectrometry was used to map the metabolome of the aging human ocular lens, and a MALDI imaging mass spectrometry method developed to quantify regional changes to the primary lens antioxidant glutathione (reduced). Spatial segmentation of total metabolite signal intensities showed the lens contains distinct metabolic compartments that change with increasing age. Using the developed absolute quantitation method, glutathione concentrations throughout the lens were mapped with up to 100 μm spatial resolution, and showed that cortical concentrations (mean = 3.7 μmol/g tissue in 34 yo) were always higher than nuclear concentrations (mean = 2.8 μmol/g tissue in 34 yo). In addition, cortical concentrations decreased slightly with increasing lens age (mean = 2.7 μmol/g tissue in 74 yo), while the decrease in nuclear glutathione concentrations was more pronounced (mean = 0.8 μmol/g tissue in 74 yo). Finally, ceramides were localised to the lens region that was specifically depleted of glutathione. In summary, MALDI FT-ICR imaging mass spectrometry has allowed age-related changes to human lens metabolites to be mapped with higher spatial resolution than previously possible using alternative techniques. In addition, the multiplex capability of MALDI imaging mass spectrometry has allowed the spatial relationships of these lens metabolites to be established and explored. This approach should aid the development of spatially directed therapies to delay the onset of lens cataract.

Purpose Lenses have an intracellular hydrostatic pressure gradient to drive fluid from central fiber cells to surface epithelial cells. Pressure is regulated by a feedback control system that relies on transient receptor potential vanilloid (TRPV)1 and TRPV4 channels. The ciliary muscle transmits tension to the lens through the zonules of Zinn. Here, we have examined if ciliary muscle tension influenced the lens intracellular hydrostatic pressure gradient. Methods We measured the ciliary body position and intracellular hydrostatic pressures in mouse lenses while pharmacologically causing relaxation or contraction of the ciliary muscle. We also used inhibitors of TRPV1 and TRPV4, in addition to phosphoinositide 3-kinase (PI3K) p110α knockout mice and immunostaining of phosphorylated protein kinase B (Akt), to determine how changes in ciliary muscle tension resulted in altered hydrostatic pressure. Results Ciliary muscle relaxation increased the distance between the ciliary body and the lens and caused a decrease in intracellular hydrostatic pressure that was dependent on intact zonules and could be blocked by inhibition of TRPV4. Ciliary contraction moved the ciliary body toward the lens and caused an increase in intracellular hydrostatic pressure and Akt phosphorylation that required intact zonules and was blocked by either inhibition of TRPV1 or genetic deletion of the p110α catalytic subunit of PI3K. Conclusions These results show that the hydrostatic pressure gradient within the lens was influenced by the tension exerted on the lens by the ciliary muscle through the zonules of Zinn. Modulation of the gradient of intracellular hydrostatic pressure in the lens could alter the water content, and the gradient of refractive index.

SUMMARYDuring development of the central nervous system oligodendrocyte precursor cells (OPCs) give rise to both myelinating oligodendrocytes and NG2 glia, which are the most proliferative cells in the adult mammalian brain. NG2 glia retain characteristics of OPCs, and some NG2 glia produce oligodendrocytes, but many others persist throughout adulthood. Why some OPCs differentiate as oligodendrocytes during development whereas others persist as OPCs and acquire characteristics of NG2 glia is not known. Using zebrafish spinal cord as a model, we found that OPCs that differentiate rapidly as oligodendrocytes and others that remain as OPCs arise in sequential waves from distinct neural progenitors. Additionally, oligodendrocyte and persistent OPC fates are specified during a defined critical period by small differences in Shh signaling and Notch activity, which modulates Shh signaling response. Thus, our data indicate that OPCs fated to produce oligodendrocytes or remain as OPCs during development are specified as distinct cell types, raising the possibility that the myelinating potential of OPCs is set by graded Shh signaling activity.

Cold temperatures can be fatal to insects, but many species have evolved the ability to cold acclimate, thereby increasing their cold tolerance. While there is a growing body of knowledge concerning the mechanisms underlying cold tolerance, relatively little is known concerning how insects sense noxious cold (cold nociception), or how cold nociception might function in cold tolerance. It has been previously shown that Drosophila melanogaster larvae perform highly stereotyped, cold-evoked behaviors under the control of noxious cold-sensing neurons (nociceptors) innervating the barrier epidermis. In the present study, we first sought to describe cold-nociceptive behavior among 11 drosophilid species with differing cold tolerances and from differing climates. Behavioral analyses revealed that the predominant cold-evoked response among drosophilid larvae is a head-to-tail contraction (CT) behavior, which is likely inherited from a common ancestor. However, despite lack of phylogenetic signal (suggesting trait lability), the CT behavior was transient and there was no clear evidence that cold sensitivity was related to thermal environment; collectively this suggests that the behavior might not be adaptive. We therefore sought to uncover an alternative way that cold nociception might be protective. Using a combination of cold-shock assays, optogenetics, electrophysiology, and methods to genetically disrupt neural transmission, we demonstrate that cold sensing neurons in Drosophila melanogaster (Class III nociceptors) are sensitized by and critical to cold acclimation. Moreover, we demonstrate that cold acclimation can be optogenetically-evoked, sans cold. Collectively, these findings reveal that cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation.Significance StatementMany insects adapt to cold in response to developmental exposure to cool temperatures. While there is a growing body of knowledge concerning the mechanisms underlying cold tolerance, it is unknown how sensory neurons might contribute. Here, we show that noxious cold sensing (cold nociception) is widely present among drosophilid larvae, and that cold-sensing neurons (Class III cold nociceptors) are necessary and sufficient drivers of cold acclimation. This suggests that cold acclimation has, at least in part, a neural basis.

Glucocorticoids (Gcs) potently inhibit inflammation, and regulate liver energy metabolism, often acting in a hypoxic environment. We now show hypoxic conditions open a specific GR cistrome, and prevent access of GR to part of the normoxic GR cistrome. Motif analysis identified enrichment of KLF4 binding sites beneath those peaks of GR binding exclusive to normoxia, implicating KLF4 as a pioneer, or co-factor under these conditions. Hypoxia reduced KLF4 expression, however, knockdown of KLF4 did not impair GR recruitment. KLF4 is a known target of microRNAs 103 and 107, both of which are induced by hypoxia. Expression of mimics to either microRNA103, or microRNA107 inhibited GR transactivation of normoxic target genes, thereby replicating the hypoxic effect. Therefore, studies in hypoxia reveal that microRNAs 103 and 107 are potent regulators of GR function. We have now identified a new pathway linking hypoxia through microRNAs 103 and 107 to regulation of GR function., Highlights • Oxygen tension profoundly affects glucocorticoid (Gc) action. • Gc target genes and regulatory elements are sensitive to oxygen tension. • MicroRNAs 103 and 107 mediate the hypoxic regulation of Gc action. • Hypoxia does not affect Gc receptor (GR) expression, modification or trafficking.

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Cataracts or clouding of the lens is the leading cause of blindness in the world. Age and diabetes are major risk factors, and with an increasing aging and diabetic population, the burden of cataracts will grow. Cataract surgery is an effective way to restore vision; however, alternatives to cataract surgery are required to reduce the looming cataract epidemic. Since it is well established that oxidative damage plays a major role in the etiology of cataracts, antioxidants have been promoted as therapies to delay and/or prevent cataracts. However, many antioxidant interventions including vitamin C have produced mixed results as anti-cataract therapies. Progress has been made towards our understanding of lens physiology and the mechanisms involved in the delivery and uptake of antioxidants to the lens which may guide future studies aimed at addressing some of the inconsistencies seen in previous animal and human studies. Of interest is the potential for vitamin C based supplements in delaying the onset of cataracts post vitrectomy which occurs in up to 80% of patients within two years. These targeted approaches are required to reduce the burden of cataract on hospitals and improve the quality of life of our aging and diabetic population.

A subset of lung adenocarcinomas is driven by the EML4‐ALK translocation. Even though ALK inhibitors in the clinic lead to excellent initial responses, acquired resistance to these inhibitors due to on‐target mutations or parallel pathway alterations is a major clinical challenge. Exploring these mechanisms of resistance, we found that EML4‐ALK cells parental or resistant to crizotinib, ceritinib or alectinib are remarkably sensitive to inhibition of CDK7/12 with THZ1 and CDK9 with alvocidib or dinaciclib. These compounds robustly induce apoptosis through transcriptional inhibition and downregulation of anti‐apoptotic genes. Importantly, alvocidib reduced tumour progression in xenograft mouse models. In summary, our study takes advantage of the transcriptional addiction hypothesis to propose a new treatment strategy for a subset of patients with acquired resistance to first‐, second‐ and third‐generation ALK inhibitors., Acquired drug resistance to ALK inhibitors prevents the effective management of lung cancer. This study shows that treatment with CDK inhibitors may be tested as an alternative upon development of resistance.

PurposeThe lens utilizes circulating fluxes of ions and water that enter the lens at both poles and exit at the equator to maintain its optical properties. We have mapped the subcellular distribution of the lens aquaporins (AQP0, 1, & 5) in these water influx and efflux zones and investigated how their membrane location is affected by changes in tension applied to the lens by the zonules.MethodsImmunohistochemistry using AQP antibodies was performed on axial sections obtained from rat lenses that had been removed from the eye and then fixed, or were fixedin situto maintain zonular tension. Zonular tension was pharmacologically modulated by applying either tropicamide (increased), or pilocarpine (decreased). AQP labelling was visualized using confocal microscopy.ResultsModulation of zonular tension had no effect on AQP1 or AQP0 labelling in either the water efflux, or influx zones. In contrast, AQP5 labelling changed from membranous to cytoplasmic in response to both mechanical and pharmacologically induced reductions in zonular tension in both the efflux zone, and anterior (but not posterior) influx zone associated with the lens sutures.ConclusionsAltering zonular tension dynamically regulates the membrane trafficking of AQP5 in the efflux and anterior influx zones to potentially change the magnitude of circulating water fluxes in the lens.

High-grade serous ovarian carcinoma is characterised by TP53 mutation and extensive chromosome instability (CIN). Because our understanding of CIN mechanisms is based largely on analysing established cell lines, we developed a workflow for generating ex vivo cultures from patient biopsies to provide models that support interrogation of CIN mechanisms in cells not extensively cultured in vitro. Here, we describe a “living biobank” of ovarian cancer models with extensive replicative capacity, derived from both ascites and solid biopsies. Fifteen models are characterised by p53 profiling, exome sequencing and transcriptomics, and karyotyped using single-cell whole-genome sequencing. Time-lapse microscopy reveals catastrophic and highly heterogeneous mitoses, suggesting that analysis of established cell lines probably underestimates mitotic dysfunction in advanced human cancers. Drug profiling reveals cisplatin sensitivities consistent with patient responses, demonstrating that this workflow has potential to generate personalized avatars with advantages over current pre-clinical models and the potential to guide clinical decision making., High-grade serous ovarian carcinoma is often associated with TP53 mutation and chromosomal instability (CIN). Here, the authors generate ex vivo cultures from biopsies and ascites of patients and perform characterization to evaluate CIN mechanisms and compare drug sensitivity with patient responses.

Purpose: The purpose of this article is to report the prognostic associations of acute retinal necrosis (ARN), determine the diagnostic utility of aqueous vs vitreous polymerase chain reaction (PCR), and describe outcomes of antiviral, corticosteroid, antithrombotic, and laser retinopexy treatment. Methods: A retrospective, nonrandomized case series was conducted. Primary outcomes included final visual acuity (VA), severe VA loss (≥1.00 log of the minimum angle of resolution [logMAR] [Snellen equivalent, Results: Fifty eyes of 46 patients were diagnosed with ARN. Varicella-zoster and herpes simplex viruses were the viral etiology in 29 and 16 eyes, respectively. Five were diagnosed on clinical criteria. Intravenous acyclovir was administered in 90% of eyes. Neither induction therapy for ≥13 days ( P = .692) nor intravitreal medication ( P = .203) decreased the incidence of severe vision loss. Maintenance antiviral medication was administered in 96% of eyes (50% received >13 weeks). At final follow-up, 40% of eyes had severe vision loss (range, –0.08 to +3.0 logMAR [Snellen, 20/17-no perception of light]). Forty-six percent developed RD. Relative afferent pupillary defect (RAPD), zone and extent of retinitis, and RD were prognostic of final VA. Antithrombotic therapy was administered in 25/50 eyes and was associated with lower incidence of severe VA loss ( P = .027) and better final VA ( P = .040). Laser retinopexy was performed in 40% of eyes with no significant effect on RD incidence ( P = .901) or severe VA loss ( P = .451). Conclusions: ARN has a high incidence of RD and poor visual outcomes. Aqueous humor PCR is the first-line diagnostic investigation. Prognostic indicators include RD, RAPD, zone, and extent of retinitis. Induction antiviral medication is appropriate for at least 12 days. Antithrombotic medication is associated with improved outcomes. Laser retinopexy does not reduce the incidence of RD.

Patient dissatisfaction after total knee arthroplasty (TKA) is a concern. Surgical error is a common, avoidable cause of failed TKA. Correct femoral and tibial component sizing improves implant longevity, clinical outcomes, knee balance, and pain scores. We hypothesized that preoperative three-dimensional (3D) templating for robot-assisted TKA (RA-TKA) is more accurate than two-dimensional (2D) digital templating. Prospectively collected data from 31 RA-TKAs were assessed to determine accuracy pertaining to implant sizing and positioning. All cases undergoing RA-TKA undergo preoperative CT-scans as per protocol. Three blinded observers retrospectively templated these knees for TKA using standard radiographs. We compared whether 2D templating was as accurate as CT-guided templating. Postoperative radiographs were then evaluated for sizing and positioning. Intraclass correlation coefficients (ICCs) and the effect of learning curve were assessed. Preoperative femoral component 3D templating and retrospective blinded 2D templating accuracies were 96.6% and 52.9%, respectively (χ 2: 17.965; odds ratio [OR]: 24.957, 3.250–191.661; p

In an era of increasing numbers of hip and knee replacements, strategies to manage prosthetic joint infection (PJI) that are effective at infection control with good patient-reported outcomes and cost containment for health systems are needed. Interest in single-stage exchange for PJI is rising and we assess evidence from the last 5 years related to this treatment strategy. Only five series for total knee replacement and ten series for total hip replacement have been reported in the last five years. More review articles and opinion pieces have been written. Reinfection rates in these recent studies range from 0 to 65%, but a meta-analysis and systematic review of all studies showed a reinfection rate of 7.6% (95% CI 3.4–13.1) and 8.8% (95% CI 7.2–10.6) for single-stage and two-stage revisions respectively. There is emerging evidence to support single-stage revision in the setting of significant bony deficiency and atypical PJIs such as fungal infections. Prospective randomised studies are recruiting and are necessary to guide the direction of single-stage revision selection criteria. The onus of surgical excellence in mechanical removal of implants, necrotic tissue, and biofilms lies with the arthroplasty surgeon and must remain the cornerstone of treatment. Single-stage revision may be considered the first-line treatment for all PJIs unless the organism is unknown, the patient is systemically septic, or there is a poor tissue envelope.

Cycads are the most endangered of plant groups based on IUCN Red List assessments; all are in Appendix I or II of CITES, about 40% are within biodiversity ‘hotspots,’ and the call for action to improve their protection is long-standing. We contend that progress in this direction will not be made until there is better understanding of cycad pollen, seed and tissue biology, which at the moment is limited to relatively few (

Total hip arthroplasty (THA) is a common surgical procedure that improves patients' quality of life and function with great and reproducible success. However, long-term pain is experienced in 7–23% of patients. This article discusses potential predisposing factors, their aetiology and the subsequent evaluation of persisting pain after THA. A thorough, structured approach to clinical history and examination allied with knowledge of potential differential diagnoses will help focus subsequent investigations to determine the cause. Diagnosis allows the provision of the most appropriate nonoperative or operative treatment course.

In previous work, we have shown the Sodium/Potassium/2 Chloride Cotransporter (NKCC1) to be a key effector of lens fiber cell volume regulation. Since others have shown that the activity of NKCC1 is regulated via its phosphorylation status, the purpose of this study was to investigate whether NKCC1 phosphorylation can be modulated in organ cultured bovine lenses, and to see how this relates to changes in lens wet weight. Western blotting was first used to confirm the expression of NKCC1, phosphorylated NKCC1 (NKCC1-P) and the regulatory kinases WNK/SPAK and phosphatases PP1/PP2A in bovine lenses at the protein level. Changes to NKCC1-P status were then assessed by organ culturing bovine lenses in either isotonic, hypertonic or hypotonic solutions in the presence or absence of the NKCC inhibitor, bumetanide, or phosphatase inhibitors okadaic acid and calyculin A. After 1–22 h of culturing, lenses were weighed, assessed for transparency and the cortical protein fractions analyzed by western blot using antibodies to detect total NKCC1 and NKCC1-P. NKCC1, NKCC1-P, SPAK, PP1 and PP2A were all detected in the membrane fraction of bovine lenses. Under hypertonic conditions, NKCC1 is phosphorylated and activated to mediate a regulatory volume increase. Finally, NKCC1-P signal increased in the presence of phosphatase inhibitors indicating that PP1/PP2A can dephosphorylate NKCC1. These results show that the phosphorylation status and hence activity of NKCC1 is dynamically regulated and that in response to hypertonic stress, NKCC1 activity is increased to effect a regulatory volume increase that limits cell shrinkage. These findings support the view that the lens dynamically regulates ion fluxes to maintain steady state lens volume, and suggest that dysfunction of this regulation maybe an initiating factor in the localized fiber cell swelling that is a characteristic of diabetic lens cataract.

Background Ulcerative Colitis (UC) is a chronic disease with periods of relapse and remission. Bowel urgency, the sudden or immediate need for a bowel movement, is one of the most bothersome symptoms experienced by patients with UC. This study explored how patients define urgency severity and what would be a meaningful improvement based on a numeric rating scale (NRS). Methods In-depth interviews were conducted in the United States with 19 adults with clinician-confirmed moderate to severe UC. Participants were asked to define levels of bowel urgency severity using an 11-point NRS (where 0 = no urgency and 10 = worst possible urgency) and to describe what would be a meaningful improvement based on how this change would impact their daily life. Interviews were audio-recorded, transcribed, and coded using thematic coding in NVivo. Results Mild bowel urgency was described as ranging between 0 to 3 on the 11-point scale by most UC patients (n=16, 84%). Mild was described as “almost normal” or “normal urgency” (n=6, 32%) and being able to make it to the bathroom with ease (n=5, 25%). Most participants (n=15, 79%) rated moderate urgency between 4 to 6 on the 11-point scale. At this level, participants commented that they would need to ensure they are close to a bathroom (n=7, 37%). Finally, ratings for severe urgency clustered between 6 to 8 (n=7, 37%) and 8 to 10 (n=12, 63%). Severe bowel urgency was described as an immediate or uncontrollable need (n=8, 42%) which impacts their ability to leave the home (n=7, 37%). When asked about what change on the urgency NRS would be meaningful, n=10 (53%) participants reported that a 1-point change would be meaningful, with most stating that any change was an improvement. Participants commented that a 1-point change would reflect improved emotional well-being and greater confidence to leave the home. In contrast, 9 (47%) participants wanted improvements of more than 1-point change; 5 (26%) indicated that a 2-point improvement was required to consider the change meaningful and 4 (21%) felt a 3-point change or more was needed. With this greater level of change, participants described having a significant improvement in urgency, with less frequent urges and improved emotional well-being. Conclusion This qualitative study revealed that UC patients largely agreed regarding ratings of ‘mild’ and ‘moderate’ bowel urgency on an 11-point severity scale. ‘Severe’ bowel urgency was split between two ranges suggesting the existence of ‘very severe’ bowel urgency. In addition, the majority of UC patients perceived a 1 or 2-point reduction on the urgency NRS as a meaningful change in their lives.

Summary Low temperatures can be fatal to insects, but many species have evolved the ability to cold acclimate, thereby increasing their cold tolerance. It has been previously shown that Drosophila melanogaster larvae perform cold-evoked behaviors under the control of noxious cold-sensing neurons (nociceptors), but it is unknown how the nervous system might participate in cold tolerance. Herein, we describe cold-nociceptive behavior among 11 drosophilid species; we find that the predominant cold-evoked larval response is a head-to-tail contraction behavior, which is likely inherited from a common ancestor, but is unlikely to be protective. We therefore tested the hypothesis that cold nociception functions to protect larvae by triggering cold acclimation. We found that Drosophilamelanogaster Class III nociceptors are sensitized by and critical to cold acclimation and that cold acclimation can be optogenetically evoked, sans cold. Collectively, these findings demonstrate that cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation., Graphical abstract, Highlights • Drosophila larvae respond to cold via behaviors which are not obviously protective • Genetically silencing cold nociceptors results in an inability to cold acclimate • Cold acclimation results in nociceptor hypersensitization • Cold tolerance can be improved by activating cold nociceptors sans cold, Biological sciences; Physiology; Neuroscience; Behavioral neuroscience; Cellular neuroscience; Sensory neuroscience

(1) To investigate whether the occlusion effect and hearing attenuation produced by 3M Combat Arms Ear Plugs (CAEP) affects balance when compared to no hearing protection and (2) to investigate whether the occlusion effect and noise-canceling capabilities of the Nacre QuietPro system affects balance when compared to no hearing protection.This prospective study collected pilot data for investigation of mechanisms of balance. 20 subjects with normal hearing and no vestibular dysfunction were tested with blackened goggles in three conditions-no hearing protection, CAEP, and with the Nacre QuietPro.A static posturogrpahy forceplate was used to measure center of gravity angular acceleration for a period of 20 seconds. The order of the conditions tested was randomized for each individual. Mean angular acceleration and standard deviation (degrees/second) of the three conditions were: (1) no hearing protection (control), 0.65 + 0.19, (2) CAEP, 0.69 + 0.23, and (3) QuietPro, 0.70 + 0.20 (one-way analysis of variance [ANOVA], df = 2, F = 0.38, p = 0.706).The components of an intact balance system include a variety of neural inputs, to include vesitbuloocular, vestibulospinal, and labyrinthine afferents. Both the CAEP and Nacre QuietPro are hearing preservation devices utilized during Operation Iraqi Freedom and Operation Enduring Freedom in Afghanistan. Our pilot data show no decrement in balance with utilization of these combat hearing preservation devices.

The optical properties of the ocular lens are important to overall vision quality. As a transparent biological tissue, the lens contributes to the overall and dynamic focussing power of the eye, and corrects for optical errors introduced by the cornea. The optical properties of the lens change throughout life. Alterations to the refractive properties and transparency of the lens result in presbyopia and cataract, respectively. However, it is not well understood how changes to lens cellular structure and function initiate these changes in refraction and transparency. Here, we attempt to bridge this knowledge gap by reviewing how the optical properties of the lens are first established, and then maintained at the cellular level throughout the lifetime of an individual. Central to this understanding is the fact that the lens has a microcirculation system that generates a flux of ions and water that circulates through the lens. By supporting ionic and metabolic homeostasis in the lens, the system actively maintains lens transparency, and by regulating the steady state water content of the lens, controls the two key parameters, lens geometry and the gradient of refractive index, which determine the refractive properties of the lens. Thus, water transport is emerging as the critical parameter that links the transparency and refractive properties of the lens at the cellular level, and highlights the need to study how age-related changes in water transport result in presbyopia and cataract, the leading causes of refractive error and blindness in the world today.

Implant malalignment may predispose patients to prosthetic failure following total knee arthroplasty (TKA). A more thorough understanding of the surgeon-specific factors that contribute to implant malalignment following TKA may uncover actionable strategies for improving implant survival. The purpose of this study was to determine the impact of surgeon volume and training status on malalignment.In this retrospective multicenter study, we performed a radiographic analysis of 1,570 primary TKAs performed at 4 private academic and state-funded centers in the U.S. and U.K. Surgeons were categorized as high-volume (≥50 TKAs/year) or low-volume (50 TKAs/year), and as a trainee (fellow/resident under the supervision of an attending surgeon) or a non-trainee (attending surgeon). On the basis of these designations, 3 groups were defined: high-volume non-trainee, low-volume non-trainee, and trainee. The postoperative medial distal femoral angle (DFA), medial proximal tibial angle (PTA), and posterior tibial slope angle (PSA) were radiographically measured. Outlier measurements were defined as follows: DFA, outside of 5° ± 3° of valgus; PTA,±3° deviation from the neutral axis; and PSA,0° or7° of flexion for cruciate-retaining or0° or5° of flexion for posterior-stabilized TKAs. "Far outliers" were defined as measurements falling± 2° outside of these ranges. The proportions of outliers were compared between the groups using univariate and multivariate analyses.When comparing the high and low-volume non-trainee groups using univariate analysis, the proportions of knees with outlier measurements for the PTA (5.3% versus 17.4%) and PSA (17.4% versus 28.3%) and the proportion of total outliers (11.8% versus 20.7%) were significantly lower in the high-volume group (all p0.001). The proportions of DFA (1.9% versus 6.5%), PTA (1.8% versus 5.7%), PSA (5.5% versus 12.6%), and total far outliers (3.1% versus 8.3%) were also significantly lower in the high-volume non-trainee group (all p0.001). Compared with the trainee group, the high-volume non-trainee group had significantly lower proportions of DFA (12.6% versus 21.6%), PTA (5.3% versus 12.0%), PSA (17.4% versus 33.3%), and total outliers (11.8% versus 22.3%) (all p0.001) as well as DFA (1.9% versus 3.9%; p = 0.027), PSA (5.5% versus 12.6%; p0.001), and total far outliers (3.1% versus 6.4%; p = 0.004). No significant differences were identified when comparing the low-volume non-trainee group and the trainee group, with the exception of PTA outliers (17.4% versus 12.0%; p = 0.041) and PTA far outliers (5.7% versus 2.6%; p = 0.033). Findings from multivariate analysis accounting for the effects of patient age, body mass index, and individual surgeon demonstrated similar results.Low surgical volume and trainee status were risk factors for outlier and far-outlier malalignment in primary TKA, even when accounting for differences in individual surgeon and patient characteristics. Trainee surgeons performed similarly, and certainly not inferiorly, to low-volume non-trainee surgeons. Even among high-volume non-trainees, the best-performing cohort in our study, the proportion of TKA alignment outliers was still high.Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Aim We compared the outcomes of transplanting expanded criteria donor (ECD) kidneys undergoing machine perfusion (MP) versus cold storage (CS). Material and methods Data on all expanded criteria deceased donor kidney transplants performed at the University of Pittsburgh Medical Center from January 2003 through December 2012 were collected from an in-house electronic repository. There were 78 patients in the MP group and 101 patients in the CS group. The majority of the ECD kidneys were imported from other organ procurement organizations: 69 of 73 in the MP group (94.5%, 5 from unknown sources); and 90 of 99 in the CS group (91%), 2 from an unknown source). Most of the patients in the MP group (77 of 78) received a combination of MP and static CS. MP was performed just prior to transplantation in all MP patients. We used descriptive statistics to characterize our sample. We used logistic regression analysis to model the binary outcome of delayed graft function (DGF; i.e., "yes/no") and Cox (proportional hazard) regression to model time until graft failure. The Kaplan-Meier product-limit method was used to estimate survival curves for graft and patient survival. Results A total of 179 transplants were done from ECD donors (MP, 78; CS, 101). The mean static cold storage time was 14 ± 4.1 hours and the mean machine perfusion time was 11.2 ± 6.3 hours in the MP group. The donor creatinine was higher (1.3 ± 0.6 mg/dl vs. 1.2 ± 0.4 mg/dl, p = 0.01) and the cold ischemia time was longer (28.9 ± 10 hours vs. 24 ± 7.9 hours, p = 0.0003) in the MP patients. There were no differences between the two groups in DGF rate (20.8% [MP] vs. 25.8% [CS], p = 0.46), six-year patient survival (74% [MP] vs. 63.2% [CS], p = 0.11), graft survival (64.3% [MP] vs. 51.5% [CS], p = 0.22), and serum creatinine levels (1.5 mg/dl vs. 1.5 mg/dl) on univariate analysis. On unadjusted analysis, MP subjects without DGF had longer graft survival compared to CS subjects with DGF (p < 0.0032) and MP subjects with DGF (p < 0.0005). MP subjects without DGF had longer death-censored graft survival compared to CS subjects with DGF (p < 0.0077) and MP subjects with DGF (p < 0.0016). However, on regression analysis, MP subjects had longer graft survival than CS subjects when DGF was not present. MP subjects without DGF had longer patient survival compared to CS subjects with DGF (p < 0.0289), on unadjusted analysis. MP subjects had a reduced risk of graft failure (hazard ratio [HR], 0.34; 95% confidence interval [CI], 0.17, 0.68) and death-censored graft failure (HR, 0.44; 95% CI, 0.19, 1.00), compared to CS subjects when DGF was not present. Conclusions Reduction of DGF rates for imported ECD kidneys is vital to optimize outcomes and increase their utilization. One strategy to decrease DGF rates may be to reduce static CS time during transportation, by utilizing a portable kidney perfusion machine.

Palmitic acid hydroxystearic acids (PAHSAs) are endogenous lipids with anti-diabetic and anti-inflammatory effects. PAHSA levels are reduced in serum and adipose tissue of insulin-resistant people and high-fat diet (HFD)-fed mice. Here, we investigated whether chronic PAHSA treatment enhances insulin sensitivity and which receptors mediate PAHSA effects. Chronic PAHSA administration in chow- and HFD-fed mice raises serum and tissue PAHSA levels ∼1.4- to 3-fold. This improves insulin sensitivity and glucose tolerance without altering body weight. PAHSA administration in chow-fed, but not HFD-fed, mice augments insulin and glucagon-like peptide (GLP-1) secretion. PAHSAs are selective agonists for GPR40, increasing Ca+2 flux, but not intracellular cyclic AMP. Blocking GPR40 reverses improvements in glucose tolerance and insulin sensitivity in PAHSA-treated chow- and HFD-fed mice and directly inhibits PAHSA augmentation of glucose-stimulated insulin secretion in human islets. In contrast, GLP-1 receptor blockade in PAHSA-treated chow-fed mice reduces PAHSA effects on glucose tolerance, but not on insulin sensitivity. Thus, PAHSAs activate GPR40, which is involved in their beneficial metabolic effects.

Onset primacy is a robust behavioural phenomenon whereby humans identify the sudden appearance of an object in the environment (onset) with greater speed and accuracy than other types of visual change, such as the sudden disappearance of an object from the environment (offset). The default mode hypothesis explains this phenomenon by postulating that onset detection is the default processing mode of the attentional system. The present study aimed to test this by investigating whether onset primacy is also reflected on a neural level, as indicated by the comparative efficiency of neural processing for onsets, using electroencephalography. It was hypothesised that this relative efficiency would be reflected in the P300 event-related potential as a greater mean amplitude with an earlier peak across temporal, parietal, and occipital regions during onset detection, compared to offset detection. This prediction was tested through a change detection task in which participants were required to identify the location of onset or offset between paired images of naturalistic scenes. Results showed that P300 amplitude and latency differed between onset and offset conditions in the anticipated directions, supporting the default mode hypothesis. These results suggest that the P300 is a neural marker of onset primacy in visual change detection.

Background: Propionibacterium acnes is a common pathogen identified in postoperative shoulder infection. It has been shown to be present in culture specimens during primary shoulder arthroplasty; however, recent work has suggested that it is most likely to be a contaminant. Our aim was to identify the potential sources of contamination in shoulder arthroplasty. Methods: Tissue swabs were obtained for microbiological analysis from consecutive patients undergoing primary shoulder arthroplasty. Routine surgical technique was maintained, and 5 specimens were taken from different sites: (1) the subdermal layer, (2) the tip of the surgeon’s glove, (3) the inside scalpel blade (used for deeper incision), (4) the forceps, and (5) the outside scalpel blade (used for the skin incision). Results: Forty patients (25 female patients and 15 male patients) were included. Thirteen (33%) of the 40 patients had at least 1 culture specimen positive for P. acnes . Two (8%) of the 25 female patients and 11 (73%) of the 15 male patients had ≥1 culture specimen positive for P. acnes . The most common site of growth of P. acnes was the subdermal layer (12 positive samples), followed by the forceps (7 positive samples), the tip of the surgeon’s glove (7 positive samples), the outside scalpel blade (4 positive samples), and the inside scalpel blade (1 positive sample). There were 27 of 75 swabs that were positive on culture for P. acnes in male patients compared with 4 of 125 swabs in female patients. Male patients had 66 times (95% confidence interval, 6 to 680 times) higher odds of having a positive culture indicating subdermal colonization compared with female patients (p < 0.001). Conclusions: P. acnes is a common contaminant of the surgical field in primary shoulder arthroplasty. The subdermal layer may be the source of this contamination, and the prevalence of P. acnes in the surgical wound may be due to the surgeon’s manipulation with gloves and instruments. Our findings are consistent with those regarding the increased rates of P. acnes bacterial load and intraoperative growth in male patients compared with female patients. Clinical Relevance: P. acnes is likely to be spread throughout the surgical field from the subdermal layer via soft-tissue handling by the surgeon and instruments. Strategies need to be utilized to minimize this contact and to reduce the chance of colonization.

A 19 year old female presents to the emergency room with right facial enlargement. Clinical examination revealed a soft tissue mass of the right buccal mucosa. Treated initially as infection, the patient later turned to clinic with now rapidly enlarging and intermittently painful mass. Computed tomography with contrast showed a low attenuated buccal mass with mild enhancement lateral to the right caudal maxilla and superior mandible. Biopsy was performed and microscopic examination showed cells with moderate pleomorphism with numerous atypical mitotic figures and occasional elongated "strap" cells with eccentric nuclei. Fluorescence in situ hybridization analysis revealed no rearrangement of the FKHR gene. The diagnosis of embryonal rhabdomyosarcoma was rendered. The patient was referred to the local children's hospital for definitive treatment.

We recently discovered a structurally novel class of endogenous lipids, branched palmitic acid esters of hydroxy stearic acids (PAHSAs), with beneficial metabolic and anti-inflammatory effects. We tested whether PAHSAs protect against colitis, which is a chronic inflammatory disease driven predominantly by defects in the innate mucosal barrier and adaptive immune system. There is an unmet clinical need for safe and well tolerated oral therapeutics with direct anti-inflammatory effects. Wild-type mice were pretreated orally with vehicle or 5-PAHSA (10 mg/kg) and 9-PAHSA (5 mg/kg) once daily for 3 days, followed by 10 days of either 0% or 2% dextran sulfate sodium water with continued vehicle or PAHSA treatment. The colon was collected for histopathology, gene expression, and flow cytometry. Intestinal crypt fractions were prepared for ex vivo bactericidal assays. Bone marrow-derived dendritic cells pretreated with vehicle or PAHSA and splenic CD4+ T cells from syngeneic mice were co-cultured to assess antigen presentation and T cell activation in response to LPS. PAHSA treatment prevented weight loss, improved colitis scores (stool consistency, hematochezia, and mouse appearance), and augmented intestinal crypt Paneth cell bactericidal potency via a mechanism that may involve GPR120. In vitro, PAHSAs attenuated dendritic cell activation and subsequent T cell proliferation and Th1 polarization. The anti-inflammatory effects of PAHSAs in vivo resulted in reduced colonic T cell activation and pro-inflammatory cytokine and chemokine expression. These anti-inflammatory effects appear to be partially GPR120-dependent. We conclude that PAHSA treatment regulates innate and adaptive immune responses to prevent mucosal damage and protect against colitis. Thus, PAHSAs may be a novel treatment for colitis and related inflammation-driven diseases.

Apolipoprotein E (ApoE) belongs to a large class of proteins that solubilize lipids for physiological transport. Humans have three different APOE alleles, APOE ε2, APOE ε3, and APOE ε4, and genetic studies identified ApoE4 as the strongest genetic risk factor for Alzheimer's disease (AD). People who are homozygous for ApoE4 (i.e., ApoE4/E4) are an order of magnitude more likely to develop late-onset AD (LOAD) than ApoE3/E3 carriers. Several differences between ApoE3 and ApoE4 may contribute to AD including the observation that ApoE4 is degraded to a greater extent than ApoE3 in the human brain. Experiments with high-temperature requirement serine peptidase A1 (HtrA1), which is found in the nervous system, demonstrate that HtrA1 is an allele-selective ApoE-degrading enzyme that degrades ApoE4 more quickly than ApoE3. This activity is specific to HtrA1, as similar assays with HtrA2 showed minimal ApoE4 proteolysis and trypsin had no preference between ApoE4 and ApoE3. HtrA1 has also been reported to cleave the tau protein (Tau) and the amyloid protein precursor (APP) to hinder the formation of toxic amyloid deposits associated with AD. Competition assays with ApoE4, ApoE3, and Tau revealed that ApoE4 inhibits Tau degradation. Thus, the identification of ApoE4 as an in vitro HtrA1 substrate suggests a potential biochemical mechanism that links ApoE4 regulation of AD proteins such as Tau.

Objective Complex local crosstalk amongst endocrine cells within the islet ensures tight coordination of their endocrine output. This is illustrated by the recent demonstration that the negative feedback control by delta cells within pancreatic islets determines the homeostatic set-point for plasma glucose during mouse postnatal development. However, the close association of islet endocrine cells that facilitates paracrine crosstalk also complicates the distinction between effects mediated directly on beta cells from indirect effects mediated via local intermediates, such as somatostatin from delta cells. Methods To resolve this problem, we generated reporter mice that allow collection of pure pancreatic delta cells along with alpha and beta cells from the same islets and generated comprehensive transcriptomes for each islet endocrine cell type. These transcriptomes afford an unparalleled view of the receptors expressed by delta, alpha and beta cells, and allow the prediction of which signal targets which endocrine cell type with great accuracy. Results From these transcriptomes, we discovered that the ghrelin receptor is expressed exclusively by delta cells within the islet, which was confirmed by fluorescent in situ hybridization and qPCR. Indeed, ghrelin increases intracellular calcium in delta cells in intact mouse islets, measured by GCaMP6 and robustly potentiates glucose-stimulated somatostatin secretion on mouse and human islets in both static and perfusion assays. In contrast, des-acyl-ghrelin at the same dose had no effect on somatostatin secretion and did not block the actions of ghrelin. Conclusions These results offer a straightforward explanation for the well-known insulinostatic actions of ghrelin. Rather than engaging beta cells directly, ghrelin engages delta cells to promote local inhibitory feedback that attenuates insulin release. These findings illustrate the power of our approach to resolve some of the long-standing conundrums with regard to the rich feedback that occurs within the islet that is integral to islet physiology and therefore highly relevant to diabetes., Graphical abstract