Protein Distribution during Human Erythroblast Enucleation In Vitro

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.     

Molecular Subtypes in Head and Neck Cancer Exhibit Distinct Patterns of Chromosomal Gain and Loss of Canonical Cancer Genes

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.     

Recent changes in endometrial cancer trends among menopausal-age US women

BackgroundChanges in endometrial cancer incidence rates after the precipitous decline in menopausal hormone therapy (MHT) use in 2002 have not been evaluated.MethodsUsing data from the Surveillance, Epidemiology, and End Results Program from 1992 to 2009 (SEER 13), we identified 63 428 incident endometrial cancer cases among women ages 20–74. We compared annual percent change (APC) in endometrial cancer incidence rates from 1992 to 2002 to rates from 2003 to 2009.ResultsIn contrast to the constant endometrial cancer rate pattern observed from 1992 to 2002 (APC 0.0%), rates increased after 2002 in women 50–74 years old (2.5%; P_{APC comparison} < 0.01). Endometrial cancer incidence increased over the entire time period among women ages 20–49 (1992–2002: 1.1%; 2003–2009: 2.1%; P_{APC comparison} = 0.21). Post-2002 increases in incidence among women ages 50–74 were specific to Type I endometrial tumors (1992–2002: ?0.6%; 2003–2009: 1.6%; P_{APC comparison} < 0.01).DiscussionThe increase in endometrial cancer incidence rates after 2002 may be related to the widespread decrease in estrogen plus progestin MHT use, which has been reported to lower endometrial cancer risk in overweight and obese women.     

Inhibition of Sphingosine Kinase-2 in a Murine Model of Lupus Nephritis

Sphingosine-1-phosphate (S1P), a potent bioactive lipid, is emerging as a central mediator in inflammation and immune responses. We have previously implicated S1P and its synthetic enzyme sphingosine kinase (SK) in inflammatory and autoimmune disorders, including inflammatory bowel disease and rheumatoid arthritis. Generation of S1P requires phosphorylation of sphingosine by SK, of which there are two isoforms. Numerous studies have implicated SK1 in immune cell trafficking, inflammation and autoimmune disorders. In this study, we set out to determine the role of SK and S1P in lupus nephritis (LN). To this end, we examined S1P and dihydro-S1P (dh-S1P) levels in serum and kidney tissues from a mouse model of LN. Interestingly dh-S1P was significantly elevated in serum and kidney tissue from LN mice, which is more readily phosphorylated by SK2. Therefore, we employed the use of the specific SK2 inhibitor, ABC294640 in our murine model of LN. Treatment with ABC294640 did not improve vascular or interstitial pathology associated with LN. However, mice treated with the SK2 inhibitor did demonstrate decreases in glomerular pathology and accumulation of B and T cells in the spleen these were not statistically different from lpr mice treated with vehicle. LN mice treated with ABC294640 did not have improved urine thromboxane levels or urine proteinuria measurements. Both S1P and dh-S1P levels in circulation were significantly reduced with ABC294640 treatment; however, dh-S1P was actually elevated in kidneys from LN mice treated with ABC294640. Together these data demonstrate a role for SKs in LN; however, they suggest that inhibition of SK1 or perhaps both SK isoforms would better prevent elevations in S1P and dh-S1P and potentially better protect against LN.     

Severe progressive scoliosis in an adult female possibly secondary thoracic surgery in childhood treated with scoliosis specific Schroth physiotherapy: Case presentation

Background

Scoliosis is a complex three-dimensional (3D) spinal deformity. Acquired scoliosis in early childhood may progress into adulthood and pose an increased risk of health problems and reduction in quality of life. In Canada, patients with scoliosis are not referred for physiotherapeutic scoliosis-specific exercises (PSSE) despite the fact that Schroth physiotherapy, a scoliosis-specific 3D posture training and exercise program, can be effective in reducing pain and improving scoliosis curves, vital capacity, and overall quality of life in scoliosis patients. This case presentation shows that indeed adult curve progression can be stopped and even reversed with scoliosis specific Schroth physiotherapy (SSSPT) in an adult patient with scoliosis.

Methods

This is a retrospective case presentation involving a 23-year-old female scoliosis patient who began an outpatient Schroth physiotherapy exercise program and was initially monitored monthly and then annually for improvement in measurements of angle of trunk rotation (ATR) and chest expansion and improvement in vital capacity measured with incentive spirometry. Photos were taken to document body image periodically throughout Schroth physiotherapy treatment. Additionally, the patient completed SRS-22 quality of life questionnaires every 2 years to evaluate daily function, pain, self-imagine, mental health, and scoliosis management satisfaction.

Results

Within one month of beginning SSSPT, the patient reported no more back pain and within 2 months, reported improved breathing. The patient also benefitted from improved chest expansion, reduced scoliosis curve angles (measured in Cobb degrees), increased vital capacity, decreased ATR, and higher SRS-22 scores. She became more active and resumed all athletic activity within 8 months of beginning Schroth physiotherapy.

Conclusions

Adult scoliosis patients are not routinely referred for PSSE in Canada, even though Schroth physiotherapy, a form of PSSE, is shown to be effective in this case presentation. The patient in this case presentation was successfully treated with Schroth physiotherapy. Long-term comprehensive Schroth physiotherapy, to help correct and maintain proper posture in all aspects of daily living, should be part of scoliosis management for adult scoliosis patients in Canada to stop and reverse curve progression and to improve overall quality of life.

     

Intrauterine Growth Restriction Alters Mouse Intestinal Architecture during Development

Infants with intrauterine growth restriction (IUGR) are at increased risk for neonatal and lifelong morbidities affecting multiple organ systems including the intestinal tract. The underlying mechanisms for the risk to the intestine remain poorly understood. In this study, we tested the hypothesis that IUGR affects the development of goblet and Paneth cell lineages, thus compromising the innate immunity and barrier functions of the epithelium. Using a mouse model of maternal thromboxane A₂-analog infusion to elicit maternal hypertension and resultant IUGR, we tested whether IUGR alters ileal maturation and specifically disrupts mucus-producing goblet and antimicrobial-secreting Paneth cell development. We measured body weights, ileal weights and ileal lengths from birth to postnatal day (P) 56. We also determined the abundance of goblet and Paneth cells and their mRNA products, localization of cellular tight junctions, cell proliferation, and apoptosis to interrogate cellular homeostasis. Comparison of the murine findings with human IUGR ileum allowed us to verify observed changes in the mouse were relevant to clinical IUGR. At P14 IUGR mice had decreased ileal lengths, fewer goblet and Paneth cells, reductions in Paneth cell specific mRNAs, and decreased cell proliferation. These findings positively correlated with severity of IUGR. Furthermore, the decrease in murine Paneth cells was also seen in human IUGR ileum. IUGR disrupts the normal trajectory of ileal development, particularly affecting the composition and secretory products of the epithelial surface of the intestine. We speculate that this abnormal intestinal development may constitute an inherent “first hit”, rendering IUGR intestine susceptible to further injury, infection, or inflammation.     

Two in one: cryptic species discovered in biological control agent populations using molecular data and crossbreeding experiments

There are many examples of cryptic species that have been identified through DNA‐barcoding or other genetic techniques. There are, however, very few confirmations of cryptic species being reproductively isolated. This study presents one of the few cases of cryptic species that has been confirmed to be reproductively isolated and therefore true species according to the biological species concept. The cryptic species are of special interest because they were discovered within biological control agent populations. Two geographically isolated populations of Eccritotarsus catarinensis (Carvalho) [Hemiptera: Miridae], a biological control agent for the invasive aquatic macrophyte, water hyacinth, Eichhornia crassipes (Mart.) Solms [Pontederiaceae], in South Africa, were sampled from the native range of the species in South America. Morphological characteristics indicated that both populations were the same species according to the current taxonomy, but subsequent DNA analysis and breeding experiments revealed that the two populations are reproductively isolated. Crossbreeding experiments resulted in very few hybrid offspring when individuals were forced to interbreed with individuals of the other population, and no hybrid offspring were recorded when a choice of mate from either population was offered. The data indicate that the two populations are cryptic species that are reproductively incompatible. Subtle but reliable diagnostic characteristics were then identified to distinguish between the two species which would have been considered intraspecific variation without the data from the genetics and interbreeding experiments. These findings suggest that all consignments of biological control agents from allopatric populations should be screened for cryptic species using genetic techniques and that the importation of multiple consignments of the same species for biological control should be conducted with caution.     

Glaucoma Surgery Calculator: Limited Additive Effect of Phacoemulsification on Intraocular Pressure in Ab Interno Trabeculectomy

PurposeTo compare intraocular pressure (IOP) reduction and to develop a predictive surgery calculator based on the results between trabectome-mediated ab interno trabeculectomy in pseudophakic patients versus phacoemulsification combined with trabectome-mediated ab interno trabeculectomy in phakic patients.MethodsThis observational surgical cohort study analyzed pseudophakic patients who received trabectome-mediated ab interno trabeculectomy (AIT) or phacoemulsification combined with AIT (phaco-AIT). Follow up for less than 12 months or neovascular glaucoma led to exclusion. Missing data was imputed by generating 5 similar but non-identical datasets. Groups were matched using Coarsened Exact Matching based on age, gender, type of glaucoma, race, preoperative number of glaucoma medications and baseline intraocular pressure (IOP). Linear regression was used to examine the outcome measures consisting of IOP and medications.ResultsOf 949 cases, 587 were included consisting of 235 AIT and 352 phaco-AIT. Baseline IOP between groups was statistically significant (p≤0.01) in linear regression models and was minimized after Coarsened Exact Matching. An increment of 1 mmHg in baseline IOP was associated with a 0.73±0.03 mmHg IOP reduction. Phaco-AIT had an IOP reduction that was only 0.73±0.32 mmHg greater than that of AIT. The resulting calculator to determine IOP reduction consisted of the formula -13.54+0.73 × (phacoemulsification yes:1, no:0) + 0.73 × (baseline IOP) + 0.59 × (secondary open angle glaucoma yes:1, no:0) + 0.03 × (age) + 0.09 × (medications).ConclusionsThis predictive calculator for minimally invasive glaucoma surgery can assist clinical decision making. Only a small additional IOP reduction was observed when phacoemulsification was added to AIT. Patients with a higher baseline IOP had a greater IOP reduction.