Visualizing the chaperone-mediated folding trajectory of the G protein ß5 ß-propeller.

The Chaperonin Containing Tailless polypeptide 1 (CCT) complex is an essential protein folding machine with a diverse clientele of substrates, including many proteins with ß-propeller domains. Here, we determine the structures of human CCT in complex with its accessory co-chaperone, phosducin-like protein 1 (PhLP1), in the process of folding Gß5, a component of Regulator of G protein Signaling (RGS) complexes. Cryoelectron microscopy (cryo-EM) and image processing reveal an ensemble of distinct snapshots that represent the folding trajectory of Gß5 from an unfolded molten globule to a fully folded ß-propeller. These structures reveal the mechanism by which CCT directs Gß5 folding through initiating specific intermolecular contacts that facilitate the sequential folding of individual ß sheets until the propeller closes into its native structure. This work directly visualizes chaperone-mediated protein folding and establishes that CCT orchestrates folding by stabilizing intermediates through interactions with surface residues that permit the hydrophobic core to coalesce into its folded state.

Visualizing the chaperone-mediated folding trajectory of the G protein ß5 ß-propeller.

The cytosolic Chaperonin Containing Tailless polypeptide 1 (CCT) complex is an essential protein folding machine with a diverse clientele of substrates, including many proteins with ß-propeller domains. Here, we determined structures of CCT in complex with its accessory co-chaperone, phosducin-like protein 1 (PhLP1), in the process of folding Gß5, a component of Regulator of G protein Signaling (RGS) complexes. Cryo-EM and image processing revealed an ensemble of distinct snapshots that represent the folding trajectory of Gß5 from an unfolded molten globule to a fully folded ß-propeller. These structures reveal the mechanism by which CCT directs Gß5 folding through initiating specific intermolecular contacts that facilitate the sequential folding of individual ß-sheets until the propeller closes into its native structure. This work directly visualizes chaperone-mediated protein folding and establishes that CCT directs folding by stabilizing intermediates through interactions with surface residues that permit the hydrophobic core to coalesce into its folded state.

The Molecular Chaperone CCT Sequesters Gelsolin and Protects it from Cleavage by Caspase-3.

The actin filament severing and capping protein gelsolin plays an important role in modulation of actin filament dynamics by influencing the number of actin filament ends. During apoptosis, gelsolin becomes constitutively active due to cleavage by caspase-3. In non-apoptotic cells gelsolin is activated by the binding of Ca2+. This activated form of gelsolin binds to, but is not a folding substrate of the molecular chaperone CCT/TRiC. Here we demonstrate that in vitro, gelsolin is protected from cleavage by caspase-3 in the presence of CCT. Cryoelectron microscopy and single particle 3D reconstruction of the CCT:gelsolin complex reveals that gelsolin is located in the interior of the chaperonin cavity, with a placement distinct from that of the obligate CCT folding substrates actin and tubulin. In cultured mouse melanoma B16F1 cells, gelsolin co-localises with CCT upon stimulation of actin dynamics at peripheral regions during lamellipodia formation. These data indicate that localised sequestration of gelsolin by CCT may provide spatial control of actin filament dynamics.

Oral octreotide capsules for the treatment of acromegaly: comparison of 2 phase 3 trial results.

PURPOSE: Results are presented from 2 to 3 trials investigating oral octreotide capsules (OOC) as an alternative to injectable somatostatin receptor ligands (iSRLs) in the treatment of acromegaly. METHODS: CH-ACM-01 was an open-label trial (N = 155) and CHIASMA OPTIMAL was a double-blind placebo-controlled (DPC) trial (N = 56), both investigating OOC as maintenance therapy for patients with acromegaly who were biochemical responders receiving iSRLs. RESULTS: Baseline characteristics in both trials reflected those expected of patients with acromegaly responding to treatment and were similar between trials, despite differences in inclusion criteria. OOC demonstrated a consistent degree of biochemical response across trials, with 65% of patients in CH-ACM-01 maintaining response during the core period and 64% of patients in CHIASMA OPTIMAL at the end of the DPC. Mean insulin-like growth factor I (IGF-I) levels remained within inclusion criteria at the end of treatment in both trials. Of 110 patients entering the fixed-dose phase in CH-ACM-01, 80% maintained or improved acromegaly symptoms from baseline to the end of treatment. Over 85% of patients in both trials elected to continue into the extension phases. OOC were found to be well tolerated across both trials, and no dose-related adverse events were observed. CONCLUSIONS: OOC demonstrated remarkably consistent results for biochemical response, durability of response, and preference to continue with oral treatment across these 2 complementary landmark phase 3 trials, despite differences in the design of each. Trial registration NCT03252353 (August 2017), NCT01412424 (August 2011).

Molecular architecture of the Bardet-Biedl syndrome protein 2-7-9 subcomplex.

Bardet-Biedl syndrome (BBS) is a genetic disorder characterized by malfunctions in primary cilia resulting from mutations that disrupt the function of the BBSome, an 8-subunit complex that plays an important role in protein transport in primary cilia. To better understand the molecular basis of BBS, here we used an integrative structural modeling approach consisting of EM and chemical cross-linking coupled with MS analyses, to analyze the structure of a BBSome 2-7-9 subcomplex consisting of three homologous BBS proteins, BBS2, BBS7, and BBS9. The resulting molecular model revealed an overall structure that resembles a flattened triangle. We found that within this structure, BBS2 and BBS7 form a tight dimer through a coiled-coil interaction and that BBS9 associates with the dimer via an interaction with the α-helical domain of BBS2. Interestingly, a BBS-associated mutation of BBS2 (R632P) is located in its α-helical domain at the interface between BBS2 and BBS9, and binding experiments indicated that this mutation disrupts the BBS2-BBS9 interaction. This finding suggests that BBSome assembly is disrupted by the R632P substitution, providing molecular insights that may explain the etiology of BBS in individuals harboring this mutation.

Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly.

The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, ß-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar ß-propeller proteins.

Budget impact of pasireotide LAR for the treatment of acromegaly, a rare endocrine disorder.

BACKGROUND: Acromegaly is a rare disorder characterized by the over-production of growth hormone (GH). Patients often experience a range of chronic comorbidities including hypertension, cardiac dysfunction, diabetes, osteoarthropathy, and obstructive sleep apnea. Untreated or inadequately controlled patients incur substantial healthcare costs, while normalization of GH levels may reduce morbidity and mortality rates to be comparable to the general population. OBJECTIVE: To assess the 3-year budget impact of pasireotide LAR on a US managed care health plan following pasireotide LAR availability. METHODS: Two separate economic models were developed: one from the perspective of an entire health plan and another from the perspective of a pharmacy budget. The total budget impact model includes costs of drug therapies and other costs for treatment, monitoring, management of adverse events, and comorbidities. The pharmacy cost calculator only considers drug costs. RESULTS: The total estimated budget impact associated with the introduction of pasireotide LAR is 0.31 cents ($0.0031) per member per month (PMPM) in the first year, 0.78 cents ($0.0078) in the second year, and 1.42 cents ($0.0142) in the third year following FDA approval. Costs were similar or lower from a pharmacy budget impact perspective. For each patient achieving disease control, cost savings from reduced comorbidities amounted to $10,240 per year. LIMITATIONS: Published data on comorbidities for acromegaly are limited. In the absence of data on acromegaly-related costs for some comorbidities, comorbidity costs for the general population were used (may be under-estimates). CONCLUSIONS: The budget impact of pasireotide LAR is expected to be modest, with an expected increase of 1.42 cents PMPM on the total health plan budget in the third year after FDA approval. The efficacy of pasireotide LAR in acromegaly, as demonstrated in head-to-head trials compared with currently available treatment options, is expected to be associated with a reduction of the prevalence of comorbidities.

Budget impact of pasireotide for the treatment of Cushing's disease, a rare endocrine disorder associated with considerable comorbidities.

OBJECTIVES: Cushing's disease (CD) is a rare condition with a prevalence of roughly 39 cases per million in the general population. Healthcare costs are substantial for CD patients with either untreated or inadequately controlled disease. This study assesses the 3-year budget impact of pasireotide on a US managed care health plan following pasireotide (Signifor) availability. METHODS: Two scenarios were evaluated to understand the differences in costs associated with the introduction of pasireotide. The first scenario evaluates the budget impact of pasireotide from the perspective of an entire health plan (total budget impact) and the second from the perspective of the pharmacy budget (pharmacy budget impact). Both scenarios evaluate the annual incremental budget impact with and without pasireotide. Scenario 1 includes costs for medical procedures, drug therapies, monitoring, surgical complications, comorbidities for patients with controlled or uncontrolled CD, and adverse events. Procedures include transsphenoidal surgery, bilateral adrenalectomy, radiotherapy and radiosurgery. Drugs include pasireotide (indicated for CD), mifepristone (indicated to control hyperglycemia secondary to hypercortisolism in patients with Cushing's syndrome) as well as several off-label treatments (ketoconazole, cabergoline, mitotane). Scenario 2 considers costs solely from the perspective of a health plan pharmacy. Costs are in $2013. RESULTS: The estimated total budget impact is $0.0115 per-member per-month (PMPM) in the first year following FDA approval, $0.0184 in the second year, and $0.0194 in the third year. Introduction of pasireotide is expected to increase the pharmacy budget by $0.0257 PMPM in the first year, $0.0363 in the second year, and $0.0360 in the third year. LIMITATIONS: Model inputs rely on the small body of literature available for Cushing's disease. CONCLUSIONS: Cushing's disease is severe disease with debilitating comorbidities and substantial healthcare costs when untreated or inadequately controlled. The inclusion of pasireotide in a health plan formulary appears to have only a small impact on the total health plan or pharmacy budget.

Delayed postoperative hyponatremia after transsphenoidal surgery: prevalence and associated factors.

OBJECT: Transient delayed postoperative hyponatremia (DPH) after transsphenoidal surgery (TSS) is common and can have potentially devastating consequences. However, the true prevalence of transient symptomatic and asymptomatic DPH has not been studied in a large patient cohort with close and accurate follow-up. METHODS: A retrospective analysis of a single-institution prospective database was conducted; all patients undergoing TSS for lesions involving the pituitary gland were followed up in a multidisciplinary neuroendocrine clinic, and demographic, imaging, and clinical data were prospectively collected. Patients were examined preoperatively and followed up postoperatively in a standardized fashion, and their postoperative sodium levels were measured at Weeks 1 and 2 postoperatively. Levels of hyponatremia were rated as mild (serum sodium concentration 130-134 mEq/L), moderate (125-129 mEq/L), or severe (< 125 mEq/L). Routine clinical questionnaires were administered at all postoperative office visits. Postoperative hyponatremia was analyzed for correlations with demographic and clinical features and with immediate postoperative physiological characteristics. RESULTS: Over a 4-year interval, 373 procedures were performed in 339 patients who underwent TSS for sellar and parasellar lesions involving the pituitary gland. The mean (± SD) age of patients was 48 ± 18 years; 61.3% of the patients were female and 46.1% were obese (defined as a body mass index [BMI] ≥ 30). The overall prevalence of DPH within the first 30 days postoperatively was 15.0%; 7.2% of the patients had mild, 3.8% moderate, and 3.8% severe hyponatremia. The incidence of symptomatic hyponatremia requiring hospitalization was 6.4%. The Fisher exact test detected a statistically significant association of DPH with female sex (p = 0.027) and a low BMI (p = 0.001). Spearman rank correlation detected a statistically significant association between BMI and nadir serum sodium concentration (r = 0.158, p = 0.002) and an inverse association for age (r = -0.113, p = 0.031). Multivariate analyses revealed a positive correlation between postoperative hyponatremia and a low BMI and a trend toward association with age; there were no associations between other preoperative demographic or perioperative risk factors, including immediate postoperative alterations in serum sodium concentration. Patients were treated with standardized protocols for hyponatremia, and DPH was not associated with permanent morbidity or mortality. CONCLUSIONS: Delayed postoperative hyponatremia was a common result of TSS; a low BMI was the only clear predictor of which patients will develop DPH. Alterations in immediate postoperative sodium levels did not predict DPH. Therefore, an appropriate index of suspicion and close postoperative monitoring of serum sodium concentration should be maintained for these patients, and an appropriate treatment should be undertaken when hyponatremia is identified.

Treatment of pituitary-dependent Cushing's disease with the multireceptor ligand somatostatin analog pasireotide (SOM230): a multicenter, phase II trial.

CONTEXT: There is currently no medical therapy for Cushing's disease that targets the pituitary adenoma. Availability of such a medical therapy would be a valuable therapeutic option for the management of this disorder. OBJECTIVE: Our objective was to evaluate the short-term efficacy of the novel multireceptor ligand somatostatin analog pasireotide in patients with de novo, persistent, or recurrent Cushing's disease. DESIGN: We conducted a phase II, proof-of-concept, open-label, single-arm, 15-d multicenter study. PATIENTS: Thirty-nine patients with either de novo Cushing's disease who were candidates for pituitary surgery or with persistent or recurrent Cushing's disease after surgery without having received prior pituitary irradiation. INTERVENTION: Patients self-administered sc pasireotide 600 microg twice daily for 15 d. MAIN OUTCOME MEASURE: Normalization of urinary free cortisol (UFC) levels after 15 d treatment was the main outcome measure. RESULTS: Of the 29 patients in the primary efficacy analysis, 22 (76%) showed a reduction in UFC levels, of whom five (17%) had normal UFC levels (responders), after 15 d of treatment with pasireotide. Serum cortisol levels and plasma ACTH levels were also reduced. Steady-state plasma concentrations of pasireotide were achieved within 5 d of treatment. Responders appeared to have higher pasireotide exposure than nonresponders. CONCLUSIONS: Pasireotide produced a decrease in UFC levels in 76% of patients with Cushing's disease during the treatment period of 15 d, with direct effects on ACTH release. These results suggest that pasireotide holds promise as an effective medical treatment for this disorder.

The adult growth hormone deficiency syndrome.

The diagnosis of the adult GH deficiency syndrome from a clinical and laboratory standpoint has been reviewed. Therapy guidelines and monitoring should focus on the patient's symptoms and IGF-1 concentrations from a laboratory standpoint. Successful patient/physician interaction depends on physician awareness of the symptoms of the deficiency syndrome and symptoms associated with therapy. Successful therapy with GH almost always results in an extremely satisfied patient, family, and physician.

Route of estrogen administration helps to determine growth hormone (GH) replacement dose in GH-deficient adults.

We prospectively studied two groups of GH-deficient patients during GH therapy based upon exposure of the liver to elevated (oral estrogen) or not elevated (endogenous or transdermal) sources of estrogen. We wondered whether higher concentrations of estrogen at the liver level (oral estrogen) might inhibit insulin-like growth factor I (IGF-I) secretion and alter exogenous GH requirements. In this study we compared GH replacement requirements in these two groups of women as well as with GH-treated adult hypopituitary males. The final GH dose was based upon maintenance IGF-I levels in the mid- to high normal range adjusted for age and sex or symptom tolerance. Each group [women taking oral estrogen (n = 12), women not taking oral estrogen (n = 13), and men (n = 12)] was similar in age and final IGF-I concentration. Women taking oral estrogen required 10.6 +/- 0.7 microg/kg x day or 867 +/- 45 microg/day GH, women not taking oral estrogen required 5.0 +/- 0.7 microg/kg x day or 424 +/- 57 microg/day, and men required 4.1 +/- 0.6 microg/kg x day or 376 +/- 49 microg/day to achieve similar IGF-I concentrations. GH requirements in men were not different from those in women not taking oral estrogen, but the GH requirements in both groups were significantly different from GH requirements in women taking oral estrogen. These observations may be useful in anticipating appropriate starting and final doses of GH in adult hypopituitary patients.

Rehabilitation of traumatic brain injury with associated visual dysfunction - A case report.

A step-by-step case report describes the rehabilitation program for visual dysfunction of a 52-year-old woman, beginning 2 years post-trauma. Methods to ameliorate such symptoms as diplopia, ocular discomfort, dry eye, inefficient saccadic movement, spasms of accommodation, poor reading comprehension and very limited stamina for near visual tasks are presented. Specific procedures are discussed and results are described and tabulated.

LIF-and IL-1 beta-mediated increases in substance P receptor mRNA in axotomized, explanted or dissociated sympathetic ganglia.

Regulation of substance P receptor (SPR) mRNA was examined in the rat sympathetic superior cervical ganglion (SCG) in vitro and in vivo after axotomy. Interleukin-1 beta (IL-1 beta) treatment of explanted ganglia elevated levels of SPR mRNA. By contrast, dissociated cultures of purified sympathetic neurons, purified fibroblasts, and purified Schwann cells each expressed only low levels of SPR mRNA, and treatment with the cytokine did not alter levels of the receptor mRNA. Treatment of Schwann cell or fibroblast cultures with leukemia inhibitory factor (LIF) also did not alter SPR mRNA. However, treatment of pure neuronal cultures with LIF significantly elevated levels of the receptor mRNA. Further, SPR mRNA increased in pure sympathetic neurons cultured in the presence of conditioned medium from IL-1 beta treated fibroblasts or Schwann cells; this effect was blocked in the presence of LIF antibody. This suggests that the stimulatory effects of IL-1 beta on SPR mRNA in explants is mediated by LIF release. Axotomy of the SCG in vivo resulted in a significant increase in LIF mRNA. Further, axotomy resulted in a significant increase in SPR mRNA, suggesting that LIF may mediate the increase in SPR mRNA. In view of the known effects of substance P (SP) on inflammatory responses, these observations suggest that coordinated expression of SP and SPR mRNA in neurons after nerve injury may participate in inflammatory and repair processes in the ganglion.

mRNAs encoding muscarinic and substance P receptors in cultured sympathetic neurons are differentially regulated by LIF or CNTF.

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have previously been shown to regulate neuronal choice of neurotransmitter. In this present study, these factors were shown to specifically and differentially regulate levels of both muscarinic (subtypes m1, m2, m3, m4, and m5) and substance P receptor (SPR) mRNAs in sympathetic neurons of the rat superior cervical ganglion (SCG) using solution hybridization/RNase protection analysis. In vivo, neonatal rat SCG expressed predominantly m2 (10.31 +/- 0.43 pg mRNA/micrograms total RNA) and some m1 (1.54 +/- 0.84 pg/microgram) muscarinic receptor mRNA, which increased developmentally to adult levels (m2 mRNA levels being 60% higher than those in neonates). By contrast, m3, m4, and m5 subtype mRNAs were much less abundant at all time points measured. A similar developmental regulation was found in dissociated SCG neurons in vitro. After 16 days in culture, m2 mRNA increased 334% to 15.76 +/- 0.68 pg/microgram, while m1 mRNA changed little (2.03 +/- 1.00 pg/microgram). However, LIF or CNTF treatment (5 ng/ml, 14 days) in sister cultures completely blocked this developmental increase. Further, LIF treatment blocked the normal muscarinic receptor-mediated increase in intracellular calcium (fura-2 imaging), indicating a functional change in receptor phenotype. By contrast, levels of SPR mRNA, which were low in untreated cultures (0.037 +/- 0.025 pg SPR mRNA/microgram total RNA), were elevated by LIF or CNTF treatment, to 0.866 +/- 0.034 pg/microgram and 0.662 +/- 0.148 pg/microgram, respectively. These observations indicate that muscarinic and SPR receptor expression are differentially regulated by the same factors in SCG neurons and that neuronal choice of receptor phenotype may be, at least in part, specifically regulated by cytokines/growth factors in the cellular milieu.

Cytokine-induced programmed death of cultured sympathetic neurons.

Programmed cell death (PCD) of sympathetic neurons is inhibited by nerve growth factor. However, factors that induce PCD of these cells are unknown. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, neuropoietic cytokines known to regulate sympathetic neuron gene expression, were examined for effects on survival of cultured sympathetic neurons. Treatment with LIF or ciliary neurotrophic factor caused neuronal death in a dose-dependent fashion. Inhibition of RNA or protein synthesis, or treatment with potassium, all of which prevent PCD after nerve growth factor deprivation, prevented LIF-induced death. The morphologic and ultrastructural characteristics of the neuronal death induced by LIF and by nerve growth factor deprivation were similar. Furthermore, LIF treatment resulted in DNA fragmentation with a characteristic "ladder" on Southern blot analysis. These observations suggest that neuron numbers may be regulated by factors which initiate PCD, as well as by factors which prevent it.

Leukemia inhibitory factor and ciliary neurotrophic factor regulate expression of muscarinic receptors in cultured sympathetic neurons.

Regulation of muscarinic receptor expression was studied in cultured sympathetic neurons of the neonatal rat superior cervical ganglion (SCG). Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF), both previously shown to regulate neurotransmitter development in cultured SCG neurons (Yamamori et al., 1989; Saadat et al., 1989), were examined for effects on receptor expression. Exposure of SCG neurons to LIF or CNTF (5 ng/ml) prevented the normal developmental increase in muscarinic receptors as measured by whole cell binding of N-methyl[3H]scopolamine. Reduction in receptor binding was detected within 2 to 4 days of treatment, with a 65-80% reduction after 16 days. Scatchard analysis demonstrated a reduction in total receptor number (Bmax) with no significant change in receptor affinity (Kd). Concentrations of 1 ng/ml of either factor reduced receptor expression with near-maximal effectiveness at doses of 10 ng/ml. The decrease in muscarinic receptors was not blocked by atropine, indicating that it was not agonist induced. Treatment with LIF or CNTF did not affect the survival of cultured neurons. Further, effects on receptor expression were reversible after discontinuance of treatment. Finally, treatment with either factor increased overall protein synthesis, indicating the integrity of cellular metabolism of cultures and hence the specificity of the decrease in muscarinic receptor number. LIF and CNTF thus regulate receptor as well as neurotransmitter development and could therefore play a role during synaptogenesis in the developing nervous system.

Effects of prism-induced, accommodative convergence stress on reading comprehension test scores.

Forty-eight binocular, non-presbyopic optometry students, unfamiliar with the purposes of the study, were provided with several matched passages of material to read in a given time sequence. Multiple choice questions were to be answered at the completion of each reading passage. These were administered in a randomly counterbalanced presentation (a b b a) utilizing either base-in prism or plano lenses in spectacle form. The results of this experiment showed a statistically significant lower comprehension rate with the base-in prism lenses in place as evidenced by fewer correct answers to the questions following the reading passages. This effect was found to be greater for the longer reading passages followed by a greater number of questions to be answered.