Palliative care use and utilization determinants among patients treated for advanced stage lung cancer care in the community and academic medical setting.

PURPOSE: Despite clinical guidelines, palliative care is underutilized during advanced stage lung cancer treatment. To inform interventions to increase its use, patient-level barriers and facilitators (i.e., determinants) need to be characterized, especially among patients living in rural areas or those receiving treatment outside academic medical centers. METHODS: Between 2020 and 2021, advanced stage lung cancer patients (n = 77; 62% rural; 58% receiving care in the community) completed a one-time survey assessing palliative care use and its determinants. Univariate and bivariate analyses described palliative care use and determinants and compared scores by patient demographic (e.g., rural vs. urban) and treatment setting (e.g., community vs. academic medical center) factors. RESULTS: Roughly half said they had never met with a palliative care doctor (49.4%) or nurse (58.4%) as part of cancer care. Only 18% said they knew what palliative care was and could explain it; 17% thought it was the same as hospice. After palliative care was distinguished from hospice, the most frequently cited reasons patients stated they would not seek palliative care were uncertainty about what it would offer (65%), concerns about insurance coverage (63%), difficulty attending multiple appointments (60%), and lack of discussion with an oncologist (59%). The most common reasons patients stated they would seek palliative care were a desire to control pain (62%), oncologist recommendation (58%), and coping support for family and friends (55%). CONCLUSION: Interventions should address knowledge and misconceptions, assess care needs, and facilitate communication between patients and oncologists about palliative care.

PAR2 promotes impaired glucose uptake and insulin resistance in NAFLD through GLUT2 and Akt interference.

BACKGROUND AND AIMS: Insulin resistance and poor glycemic control are key drivers of the development of NAFLD and have recently been shown to be associated with fibrosis progression in NASH. However, the underlying mechanisms involving dysfunctional glucose metabolism and relationship with NAFLD/NASH progression remain poorly understood. We set out to determine whether protease-activated receptor 2 (PAR2), a sensor of extracellular inflammatory and coagulation proteases, links NAFLD and NASH with liver glucose metabolism. APPROACH AND RESULTS: Here, we demonstrate that hepatic expression of PAR2 increases in patients and mice with diabetes and NAFLD/NASH. Mechanistic studies using whole-body and liver-specific PAR2-knockout mice reveal that hepatic PAR2 plays an unexpected role in suppressing glucose internalization, glycogen storage, and insulin signaling through a bifurcating Gq -dependent mechanism. PAR2 activation downregulates the major glucose transporter of liver, GLUT2, through Gq -MAPK-FoxA3 and inhibits insulin-Akt signaling through Gq -calcium-CaMKK2 pathways. Therapeutic dosing with a liver-homing pepducin, PZ-235, blocked PAR2-Gq signaling and afforded significant improvements in glycemic indices and HbA1c levels in severely diabetic mice. CONCLUSIONS: This work provides evidence that PAR2 is a major regulator of liver glucose homeostasis and a potential target for the treatment of diabetes and NASH.

Amifostine and rituximab in refractory immune thrombocytopaenia: A case series.

BACKGROUND AND OBJECTIVES: Management of refractory immune thrombocytopaenia (ITP) can be challenging. Amifostine, a thiophosphate prodrug, induces megakaryocyte maturation. In 2010, Fan et al. published results for 21 Chinese splenectomized patients, aged 13-92, with steroid-refractory ITP. Nineteen patients (15 patients aged >18 years) achieved remission 2 months post-amifostine. This is the first publication utilizing amifostine and rituximab in refractory ITP. MATERIALS AND METHODS: At the Cairns Hospital in Australia, we identified five patients treated with amifostine and rituximab for refractory ITP. Amifostine IV 400 mg once daily was administered 5 days/week for 5 weeks as tolerated. Rituximab was administered concurrently with/preceding amifostine based on clinician preference. Data were obtained through medical records and follow-up serology up to 5 years post-amifostine was available. RESULTS: Three cases demonstrated durable responses up to 5 years' follow-up. One patient initially achieved remission but relapsed 1-year post-amifostine. One patient who did not have a splenectomy prior to amifostine did not respond. CONCLUSION: Three out of five patients achieved durable responses with amifostine and rituximab. Although there is confounding by rituximab, given its established low durable response rate, it is likely that the excellent long-term responses achieved were a result of amifostine. Clinical trials with larger patient cohorts and further investigation are required to confirm the efficacy and mechanism of amifostine in ITP.

PAR2 controls cholesterol homeostasis and lipid metabolism in nonalcoholic fatty liver disease.

OBJECTIVE: Increases in hepatic and plasma cholesterol occur in patients with nonalcoholic fatty liver disease (NAFLD), although the reason for this is not well understood. We investigated whether Protease-Activated Receptor 2 (PAR2) plays a role in cholesterol and lipid homeostasis in NAFLD. METHODS: Human liver biopsies (n = 108) were quantified for PAR2 expression from NAFLD cases randomly selected and stratified by liver fibrosis stage, the primary predictor for clinical outcomes, while controlling for age, gender, and BMI between fibrosis groups. Demographic data and laboratory studies on plasma samples were obtained within 6 months of liver biopsy. Wild-type and PAR2-KO (C57BL/6 F2rl1-/-) mice were fed either normal or high fat diet for 16 weeks and plasma and liver assayed for lipids and soluble metabolites. RESULTS: Severity of NAFLD and plasma cholesterol levels significantly correlated with hepatocyte PAR2 expression in NAFLD patients. Conversely, PAR2 deficiency in mice resulted in reduced expression of key hepatic genes involved in cholesterol synthesis, a 50% drop in plasma and total liver cholesterol, and induced a reverse cholesterol transport system that culminated in 25% higher fecal bile acid output. PAR2-deficient mice exhibited enhanced fatty acid ß-oxidation with a ketogenic shift and an unexpected increase in liver glycogenesis. Mechanistic studies identified Gi-Jnk1/2 as key downstream effectors of protease-activated PAR2 in the regulation of lipid and cholesterol homeostasis in liver. CONCLUSIONS: These data indicate that PAR2 may be a new target for the suppression of plasma cholesterol and hepatic fat accumulation in NAFLD and related metabolic conditions.

Outcomes in surgically resectable oropharynx cancer treated with transoral robotic surgery versus definitive chemoradiation.

PURPOSE: Optimal treatment strategies for the management of oropharyngeal squamous cell carcinoma (OPSCC) remain unclear. The objective of this study is to examine the role of transoral robotic surgery (TORS) on functional and treatment outcomes. MATERIALS AND METHODS: A retrospective review of patients with OPSCC (tonsil/base of tongue) who underwent TORS with neck dissection± adjuvant therapy between January 2011 to December 2016 were compared to a stage matched cohort of patients treated with primary chemoradiation. Demographic, treatment, and outcome data were collected. RESULTS: 54 patients received primary chemoradiation and 65 patients (surgical group) received TORS ± adjuvant therapy for clinically staged disease meeting study criteria. 25% (N = 17) were treated with surgery alone. The remainder of the surgical group received postoperative radiation (N = 48), half of which received adjuvant chemotherapy (N = 24) in addition to radiation. 63% (N = 41) of the patients did not have risk factors for chemotherapy. No differences in overall or disease free survival were observed with TORS compared to chemoradiation (p = 0.9), although Charlson Comorbidity Index (CCI) was higher in the surgical group (p = 0.01). The strongest predictor of prolonged gastrostomy tube use was not treatment, but rather co-morbidity (p = 0.03), with no significant differences beyond 12 months. CONCLUSION: Although no significant survival differences were observed across treatment groups, this was maintained despite increased comorbidity index in the surgical patients. Given the ability to de-escalate and/or eliminate adjuvant therapy, particularly in a less healthy population, TORS would appear to be the viable treatment option it has become.

Comparison of outcomes of stereotactic body radiation therapy delivered with three different technologies to the lung.

PURPOSE/OBJECTIVES: Since the inception of stereotactic body radiation therapy (SBRT), treatment delivery has been performed with volumetric modulated arc therapy (VMAT), helical tomotherapy (HT) and noncoplanar static fields (SF). The purpose of this study is to compare SBRT delivery among these treatment modalities to the lung. MATERIALS/METHODS: A retrospective review of SBRT treatments of 30 to 60 Gy in 1 to 5 fractions from 2007 to 2015 was performed. Dosimetric parameters included V5, V20, D2cm, gross tumor volume (GTV) and planning target volume (PTV) size and coverage, rib/esophageal minimum/maximum doses, R30Gy, R50%, and the conformality index (CI). Clinical outcomes evaluated included local control, pneumonitis and other toxicities. ANOVA, Student's t-test and Kruskal-Wallis test were used to compare the parameters among modalities. Kaplan-Meier estimates of time-to-local failure were produced. RESULTS: 176 Treatments included 106 SF, 36 VMAT and 34 HT. HT had better PTV coverage (p=0.0166) but higher lung V5 and esophageal doses (p<0.001 and p=0.0032). R30Gy, R50%, and CI were significantly better with VMAT SBRT (p<0.001). Clinically, Grade 2+ pneumonitis was associated with larger median GTV's of 21.39 cc versus 7.65 cc (p=0.0016), larger median PTV's of 65.62 cc versus 31.75 cc (p=0.0030), and higher V20 6.62% versus 4.08% (p=0.0408). For patients surviving >1 year, overall local failure rate was 9.4%. Actuarial control rates trended toward statistical significance with time to local failure with VMAT being the most favorable group on the Kaplan-Meier curve (p=0.0733). CONCLUSION: VMAT showed superior conformality compared to the other modalities. Among the modalities examined, HT had higher values for parameters associated with toxicity such as V5 and maximum esophageal dose, but all were within acceptable limits. There was a trend to better local control with VMAT.

Noncanonical Matrix Metalloprotease 1-Protease-Activated Receptor 1 Signaling Drives Progression of Atherosclerosis.

OBJECTIVE: Protease-activated receptor-1 (PAR1) is classically activated by thrombin and is critical in controlling the balance of hemostasis and thrombosis. More recently, it has been shown that noncanonical activation of PAR1 by matrix metalloprotease-1 (MMP1) contributes to arterial thrombosis. However, the role of PAR1 in long-term development of atherosclerosis is unknown, regardless of the protease agonist. APPROACH AND RESULTS: We found that plasma MMP1 was significantly correlated (R=0.33; P=0.0015) with coronary atherosclerotic burden as determined by angiography in 91 patients with coronary artery disease and acute coronary syndrome undergoing cardiac catheterization or percutaneous coronary intervention. A cell-penetrating PAR1 pepducin, PZ-128, currently being tested as an antithrombotic agent in the acute setting in the TRIP-PCI study (Thrombin Receptor Inhibitory Pepducin-Percutaneous Coronary Intervention), caused a significant decrease in total atherosclerotic burden by 58% to 70% (P<0.05) and reduced plaque macrophage content by 54% (P<0.05) in apolipoprotein E-deficient mice. An MMP1 inhibitor gave similar beneficial effects, in contrast to the thrombin inhibitor bivalirudin that gave no improvement on atherosclerosis end points. Mechanistic studies revealed that inflammatory signaling mediated by MMP1-PAR1 plays a critical role in amplifying tumor necrosis factor α signaling in endothelial cells. CONCLUSIONS: These data suggest that targeting the MMP1-PAR1 system may be effective in tamping down chronic inflammatory signaling in plaques and halting the progression of atherosclerosis.

Update of a Prospective Study of Stereotactic Body Radiation Therapy for Post-Chemoradiation Residual Disease in Stage II/III Non-Small Cell Lung Cancer.

PURPOSE: To report long-term outcomes (risk of late toxicities, local control, and survival) of dose escalation by stereotactic radiation therapy boost to residual fluorodeoxyglucose positron emission tomography-positive residual disease after chemoradiation (CRT) in stage III non-small cell lung cancer (NSCLC). METHODS AND MATERIALS: Patients with stage IIB/III NSCLC underwent computed tomography or positron emission tomography-computed tomography screening approximately 1 month after completion of CRT. Limited residual disease (≤5 cm) within the site of the primary tumor received a stereotactic radiation therapy boost of either 10 Gy × 2 fractions or 6.5 Gy × 3 fractions to the primary tumor, to achieve a total Biologically Equivalent Dose >100 Gy. RESULTS: Thirty-seven patients received protocol therapy. With a median follow-up of 25.2 months, the crude local control rate for the entire group was 78% (n=29), but 10 patients (29%) and 24 patients (65%) developed regional and metastatic disease, respectively. At last follow-up, 5 patients (13.5%) remain alive, all with no evidence of disease, whereas 27 (73%) died of disease and the remaining 5 (13.5%) died of other causes. Median overall survival (OS) for the entire group was 25.2 months. Predictors for grade 3 pneumonitis included age and mean lung dose. Poorer median OS was associated with histology: median OS 15.6 months for squamous cell versus 34.8 months for other histologies (large cell neuroendocrine tumors excluded) (P=.04). The median progression-free survival was 6 months, with IIIB disease having significantly worse median progression-free survival (stages IIB/IIA being 9.4 months, vs 4.7 months for stage IIIB [P=.03]). CONCLUSIONS: Stereotactic radiation therapy boost after CRT is a safe treatment resulting in improvements in local control for locally advanced NSCLC. No additional late toxicities were seen. Possible improvement in OS was found, but further study in a larger prospective trial is needed.

Frameless image-guided radiosurgery for trigeminal neuralgia.

BACKGROUND: Frameless image-guided radiosurgery (IGRS) is a safe and effective noninvasive treatment for trigeminal neuralgia (TN). This study evaluates the use of frameless IGRS to treat patients with refractory TN. METHODS: We reviewed the records of 20 patients diagnosed with TN who underwent frameless IGRS treatments between March 2012 and December 2013. Facial pain was graded using the Barrow Neurological Institute (BNI) scoring system. The initial setup uncertainty from simulation to treatment and the patient intrafraction uncertainty were measured. The median follow-up was 32 months. RESULTS: All patients' pain was BNI Grade IV or V before the frameless IGRS treatment. The mean intrafraction shift was 0.43 mm (0.28-0.76 mm), and the maximum intrafraction shift was 0.95 mm (0.53-1.99 mm). At last follow-up, 8 (40%) patients no longer required medications (BNI 1 or 2), 11 (55%) patients were pain free but required medication (BNI 3), and 1 (5%) patient had no pain relief (BNI 5). Patients who did not have prior surgery had a higher odds ratio for pain relief compared to patients who had prior surgery (14.9, P = 0.0408). CONCLUSIONS: Frameless IGRS provides comparable dosimetric and clinical outcomes to frame-based SRS in a noninvasive fashion for patients with medically refractory TN.

Targeting Liver Fibrosis with a Cell-penetrating Protease-activated Receptor-2 (PAR2) Pepducin.

Chronic liver inflammation and fibrosis in nonalcoholic steatohepatitis can lead to cirrhosis and liver failure for which there are currently no approved treatments. Protease-activated receptor-2 (PAR2) is an emerging new target expressed on liver stellate cells and hepatocytes that regulates the response to liver injury and inflammation. Here, we identified a pepducin to block the deleterious actions of PAR2 in promoting liver fibrosis. Non-alcoholic fatty liver disease and early fibrosis were induced by the methionine-choline-deficient diet in mice. Fibrotic liver disease was induced by administering carbon tetrachloride for 8 weeks. Mice were treated with the pepducin PZ-235 either from onset of the experiment or after fibrosis was established. Hepatic fibrosis, collagen content, inflammatory cytokines, steatosis, triglycerides, and NAFLD activity score were assessed as primary outcome parameters depending on the model. The activity of the PAR2 pepducin on cultured stellate cell activation and hepatocyte reactive oxygen species production was evaluated. PZ-235 significantly suppressed liver fibrosis, collagen deposition, inflammatory cytokines, NAFLD activity score, steatosis, triglycerides, aspartate transaminase, alanine transaminase, and stellate cell proliferation by up to 50-100%. The PAR2 inhibitor afforded significant protective effects against hepatocellular necrosis and attenuated PAR2-mediated reactive oxygen species production in hepatocytes. PZ-235 was distributed to liver and other mouse tissues and was found to form a well structured α-helix that closely resembles the juxtamembrane helical region of the analogous TM6 and third intracellular region of the intact receptor that is critical for coupling to internal G proteins. The ability of PZ-235 to effectively suppress fibrosis, hepatocellular necrosis, reactive oxygen species production, steatosis, and inflammation indicates the potential for PAR2 pepducin inhibitors to be broadly efficacious in the treatment of liver fibrosis.

Dexamethasone administration during definitive radiation and temozolomide renders a poor prognosis in a retrospective analysis of newly diagnosed glioblastoma patients.

BACKGROUND: Dexamethasone (DXM) is commonly used in the management of cerebral edema in patients diagnosed with glioblastoma multiforme (GBM). Bevacizumab (BEV) is FDA-approved for the progression or recurrence of GBM but has not been shown to improve survival when given for newly diagnosed patients concurrently with radiation (RT) and temozolomide (TMZ). Both DXM and BEV reduce cerebral edema, however, DXM has been shown to induce cytokine cascades which could interfere with cytotoxic therapy. We investigated whether DXM would reduce survival of GBM patients in the setting of concurrent TMZ and BEV administration. METHODS: We reviewed the treatment of all 73 patients with GBM who received definitive therapy at our institution from 2005 to 2013 with RT (60 Gy) delivered with concurrent daily TMZ (75 mg/m(2)). Of these, 34 patients also were treated with concurrent BEV (10 mg/kg every two weeks). Patients received adjuvant therapy (TMZ or TMZ/Bev) until either progression, discontinuation due to toxicity, or 12 months after radiation completion. All patients who had GBM progression with TMZ were offered BEV for salvage therapy, with 19 (56 %) receiving BEV. RESULTS: With a median follow-up of 15.6 months, 67 (91.8 %) patients were deceased. The OS for the entire cohort was 15.9 months, while the PFS was 7.7 months. The extent of resection was a prognostic indicator for OS (p = .0044). The median survival following gross tumor resection (GTR) was 22.5 months, subtotal resection (STR) was 14.9 months, and biopsy was 12.1 months. The addition of BEV to TMZ with RT was borderline significantly associated with increased PFS (9.4 vs. 5.1 months, p = 0.0574) although was not significantly associated with OS (18.1 vs. 15.3 months respectively, p = 0.3064). In patients receiving TMZ, DXM use concurrent with RT was a poor prognostic indicator of both OS (12.7 vs. 22.6 months, p = 0.003) and PFS (3.6 vs. 8.4 months, p <0.0001). DXM did not reduce OS in patients who received TMZ and BEV concurrently with RT (22.9 vs 22.8 months, p = 0.4818). On multivariable analysis, DXM use predicted an unfavorable OS hazard ratio (HR) = 1.72, p = 0.045). CONCLUSIONS: Our results with TMZ, BEV, and RT are similar to previous studies in terms of PFS and OS. DXM use during RT with concurrent TMZ correlated with reduced OS and PFS unless BEV was administered.

Mutagenesis of the aquaporin 4 extracellular domains defines restricted binding patterns of pathogenic neuromyelitis optica IgG.

Neuromyelitis optica-immunoglobulin G (NMO-IgG) binds to aquaporin-4 (AQP4) water channels in the central nervous system leading to immune-mediated injury. We have previously demonstrated that a high proportion of CSF plasma cells of NMO patients produce antibody to the extracellular domains of the AQP4 protein and that recombinant IgG (rAb) derived from these cells recapitulate pathogenic features of disease. We performed a comprehensive mutational analysis of the three extracellular loops of the M23 isoform of human AQP4 using both serial and single point mutations, and we evaluated the effects on binding of NMO AQP4-reactive rAbs by quantitative immunofluorescence. Whereas all NMO rAbs required conserved loop C ((137)TP(138) and Val(150)) and loop E ((230)HW(231)) amino acids for binding, two broad patterns of NMO-IgG recognition could be distinguished based on differential sensitivity to loop A amino acid changes. Pattern 1 NMO rAbs were insensitive to loop A mutations and could be further discriminated by differential sensitivity to amino acid changes in loop C ((148)TM(149) and His(151)) and loop E (Asn(226) and Glu(228)). Alternatively, pattern 2 NMO rAbs showed significantly reduced binding following amino acid changes in loop A ((63)EKP(65) and Asp(69)) and loop C (Val(141), His(151), and Leu(154)). Amino acid substitutions at (137)TP(138) altered loop C conformation and abolished the binding of all NMO rAbs and NMO-IgG, indicating the global importance of loop C conformation to the recognition of AQP4 by pathogenic NMO Abs. The generation of human NMO rAbs has allowed the first high resolution mapping of extracellular loop amino acids critical for NMO-IgG binding and identified regions of AQP4 extracellular structure that may represent prime targets for drug therapy.

Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency.

Extraocular muscles (EOMs) are highly specialized skeletal muscles that originate from the head mesoderm and control eye movements. EOMs are uniquely spared in Duchenne muscular dystrophy and animal models of dystrophin deficiency. Specific traits of myogenic progenitors may be determinants of this preferential sparing, but very little is known about the myogenic cells in this muscle group. While satellite cells (SCs) have long been recognized as the main source of myogenic cells in adult muscle, most of the knowledge about these cells comes from the prototypic limb muscles. In this study, we show that EOMs, regardless of their distinctive Pax3-negative lineage origin, harbor SCs that share a common signature (Pax7(+), Ki67(-), Nestin-GFP(+), Myf5(nLacZ+), MyoD-positive lineage origin) with their limb and diaphragm somite-derived counterparts, but are remarkably endowed with a high proliferative potential as revealed in cell culture assays. Specifically, we demonstrate that in adult as well as in aging mice, EOM SCs possess a superior expansion capacity, contributing significantly more proliferating, differentiating and renewal progeny than their limb and diaphragm counterparts. These robust growth and renewal properties are maintained by EOM SCs isolated from dystrophin-null (mdx) mice, while SCs from muscles affected by dystrophin deficiency (i.e., limb and diaphragm) expand poorly in vitro. EOM SCs also retain higher performance in cell transplantation assays in which donor cells were engrafted into host mdx limb muscle. Collectively, our study provides a comprehensive picture of EOM myogenic progenitors, showing that while these cells share common hallmarks with the prototypic SCs in somite-derived muscles, they distinctively feature robust growth and renewal capacities that warrant the title of high performance myo-engines and promote consideration of their properties for developing new approaches in cell-based therapy to combat skeletal muscle wasting.

Ancestral Myf5 gene activity in periocular connective tissue identifies a subset of fibro/adipogenic progenitors but does not connote a myogenic origin.

Extraocular muscles (EOM) represent a unique muscle group that controls eye movements and originates from head mesoderm, while the more typically studied body and limb muscles are somite-derived. Aiming to investigate myogenic progenitors (satellite cells) in EOM versus limb and diaphragm of adult mice, we have been using flow cytometry in combination with myogenic-specific Cre-loxP lineage marking for cell isolation. While analyzing cells from the EOM of mice that harbor Myf5(Cre)-driven GFP expression, we identified in addition to the expected GFP(+) myogenic cells (presumably satellite cells), a second dominant GFP(+) population distinguished as being Sca1(+), non-myogenic, and exhibiting a fibro/adipogenic potential. This unexpected population was not only unique to EOM compared to the other muscles but also specific to the Myf5(Cre)-driven reporter when compared to the MyoD(Cre) driver. Histological studies of periocular tissue preparations demonstrated the presence of Myf5(Cre)-driven GFP(+) cells in connective tissue locations adjacent to the muscle masses, including cells in the vasculature wall. These vasculature-associated GFP(+) cells were further identified as mural cells based on the presence of the specific XLacZ4 transgene. Unlike the EOM satellite cells that originate from a Pax3-negative lineage, these non-myogenic Myf5(Cre)-driven GFP(+) cells appear to be related to cells of a Pax3-expressing origin, presumably derived from the neural crest. In all, our lineage tracing based on multiple reporter lines has demonstrated that regardless of common ancestral expression of Myf5, there is a clear distinction between periocular myogenic and non-myogenic cell lineages according to their mutually exclusive antecedence of MyoD and Pax3 gene activity.

Sorghum mutant RG displays antithetic leaf shoot lignin accumulation resulting in improved stem saccharification properties.

BACKGROUND: Improving saccharification efficiency in bioenergy crop species remains an important challenge. Here, we report the characterization of a Sorghum (Sorghum bicolor L.) mutant, named REDforGREEN (RG), as a bioenergy feedstock. RESULTS: It was found that RG displayed increased accumulation of lignin in leaves and depletion in the stems, antithetic to the trend observed in wild type. Consistent with these measurements, the RG leaf tissue displayed reduced saccharification efficiency whereas the stem saccharification efficiency increased relative to wild type. Reduced lignin was linked to improved saccharification in RG stems, but a chemical shift to greater S:G ratios in RG stem lignin was also observed. Similarities in cellulose content and structure by XRD-analysis support the correlation between increased saccharification properties and reduced lignin instead of changes in the cellulose composition and/or structure. CONCLUSION: Antithetic lignin accumulation was observed in the RG mutant leaf-and stem-tissue, which resulted in greater saccharification efficiency in the RG stem and differential thermochemical product yield in high lignin leaves. Thus, the red leaf coloration of the RG mutant represents a potential marker for improved conversion of stem cellulose to fermentable sugars in the C4 grass Sorghum.

Comparative feedstock analysis in Setaria viridis L. as a model for C4 bioenergy grasses and Panicoid crop species.

Second generation feedstocks for bioethanol will likely include a sizable proportion of perennial C4 grasses, principally in the Panicoideae clade. The Panicoideae contain agronomically important annual grasses including Zea mays L. (maize), Sorghum bicolor (L.) Moench (sorghum), and Saccharum officinarum L. (sugar cane) as well as promising second generation perennial feedstocks including Miscanthus×giganteus and Panicum virgatum L. (switchgrass). The underlying complexity of these polyploid grass genomes is a major limitation for their direct manipulation and thus driving a need for rapidly cycling comparative model. Setaria viridis (green millet) is a rapid cycling C4 panicoid grass with a relatively small and sequenced diploid genome and abundant seed production. Stable, transient, and protoplast transformation technologies have also been developed for Setaria viridis making it a potentially excellent model for other C4 bioenergy grasses. Here, the lignocellulosic feedstock composition, cellulose biosynthesis inhibitor response and saccharification dynamics of Setaria viridis are compared with the annual sorghum and maize and the perennial switchgrass bioenergy crops as a baseline study into the applicability for translational research. A genome-wide systematic investigation of the cellulose synthase-A genes was performed identifying eight candidate sequences. Two developmental stages; (a) metabolically active young tissue and (b) metabolically plateaued (mature) material are examined to compare biomass performance metrics.

Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells.

Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing, and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.

The depletion of skeletal muscle satellite cells with age is concomitant with reduced capacity of single progenitors to produce reserve progeny.

Satellite cells are myogenic progenitors that reside on the myofiber surface and support skeletal muscle repair. We used mice in which satellite cells were detected by GFP expression driven by nestin gene regulatory elements to define age-related changes in both numbers of satellite cells that occupy hindlimb myofibers and their individual performance. We demonstrate a reduction in satellite cells per myofiber with age that is more prominent in females compared to males. Satellite cell loss also persists with age in myostatin-null mice regardless of increased muscle mass. Immunofluorescent analysis of isolated myofibers from nestin-GFP/Myf5(nLacZ/+) mice reveals a decline with age in the number of satellite cells that express detectable levels of betagal. Nestin-GFP expression typically diminishes in primary cultures of satellite cells as myogenic progeny proliferate and differentiate, but GFP subsequently reappears in the Pax7(+) reserve population. Clonal analysis of sorted GFP(+) satellite cells from hindlimb muscles shows heterogeneity in the extent of cell density and myotube formation among colonies. Reserve cells emerge primarily within high-density colonies, and the number of clones that produce reserve cells is reduced with age. Thus, satellite cell depletion with age could be attributed to a reduced capacity to generate a reserve population.

Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid.

OBJECTIVE: Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B-cell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic. METHODS: We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy- and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections. RESULTS: Whereas humanized control rAbs derived from anti-myelin hybridomas and anti-myelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immunoreactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells. INTERPRETATION: The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein.

Activation of wingless targets requires bipartite recognition of DNA by TCF.

Specific recognition of DNA by transcription factors is essential for precise gene regulation. In Wingless (Wg) signaling in Drosophila, target gene regulation is controlled by T cell factor (TCF), which binds to specific DNA sequences through a high mobility group (HMG) domain. However, there is considerable variability in TCF binding sites, raising the possibility that they are not sufficient for target location. Some isoforms of human TCF contain a domain, termed the C-clamp, that mediates binding to an extended sequence in vitro. However, the significance of this extended sequence for the function of Wnt response elements (WREs) is unclear. In this report, we identify a cis-regulatory element that, to our knowledge, was previously unpublished. The element, named the TCF Helper site (Helper site), is essential for the activation of several WREs. This motif greatly augments the ability of TCF binding sites to respond to Wg signaling. Drosophila TCF contains a C-clamp that enhances in vitro binding to TCF-Helper site pairs and is required for transcriptional activation of WREs containing Helper sites. A genome-wide search for clusters of TCF and Helper sites identified two new WREs. Our data suggest that DNA recognition by fly TCF occurs through a bipartite mechanism, involving both the HMG domain and the C-clamp, which enables TCF to locate and activate WREs in the nucleus.