Epithelial Carcinomas Arising within Phyllodes Tumours of the Breast: A Review of Their Pathological Characteristics.

Epithelial proliferation is a common feature of phyllodes tumours (PTs), but epithelial malignancy is rare. This review seeks to further our understanding of epithelial malignancy within PTs by analysing their histopathological characteristics in previously reported cases and providing an overview of studies on their pathological features. PubMed and DeepDyve were searched for case reports, case series, and literature reviews of in situ and invasive carcinoma within PTs. Only cases where the carcinoma was within the PT were included. Cases of synchronous carcinoma in the ipsilateral or contralateral breast were excluded. Ninety-eight cases of in situ or invasive carcinoma within a PT were identified. Across the grades of PTs, there was a similar proportion of invasive carcinomas compared to in situ lesions. Malignant PT correlates with a higher likelihood of epithelial malignancy, and molecular studies support a possible causal pathophysiological relationship. This higher likelihood may suggest interactions between malignant stroma and the transforming epithelium that could potentially play a significant role in the phenomenon, which remains to be elucidated. Encasement within a PT likely improves the prognosis of breast carcinoma due to earlier detection. The presence of carcinoma within a malignant PT has uncertain prognostic implications. Thorough sampling of all PTs is recommended for appropriate prognostication and management.

Intra-operative extracorporeal irradiation of tumour-invaded craniotomy bone flap in meningioma: a case series.

BACKGROUND: Extracorporeal irradiation of tumorous calvaria (EITC) can be performed to restore function and form of the skull after resection of bone-invasive meningioma. We sought to examine the rate of tumour recurrence and other selected outcomes in patients undergoing meningioma resection and EITC. METHODS: Retrospective single-centre study of adult patients undergoing meningioma resection and EITC between January 2015 and November 2022 at a tertiary neurosurgical centre. Patient demographics, surgery data, tumour data, use of adjuvant therapy, surgical complications, and tumour recurrences were collected. RESULTS: Eighteen patients with 11 (61%) CNS WHO grade 1, 6 (33%) grade 2, and 1 (6%) grade 3 meningiomas were included. Median follow-up was 42 months (range 3-88). Five (28%) patients had a recurrence, but none were associated with the bone flap. Two (11%) wound infections requiring explant surgery occurred. Six (33%) patients required a further operation. Two operations were for recurrences, one was for infection, one was a washout and wound exploration but no evidence of infection was found, one patient requested the removal of a small titanium implant, and one patient required a ventriculoperitoneal shunt for a persistent CSF collection. There were no cases of bone flap resorption and cosmetic outcome was not routinely recorded. CONCLUSION: EITC is feasible and fast to perform with good outcomes and cost-effectiveness compared to other reconstructive methods. We observed similar recurrence rates and lower infection rates requiring explant compared to the largest series of cranioplasty in meningioma. Cosmetic outcome is universally under-reported and should be reported in future studies.

Key Moments in the History of Liposuction by Dermatologists.

Dermatologists have been pioneers in the development and refinement of liposuction using local anesthesia. Although other specialties routinely use general anesthesia for liposuction, the safety profile of liposuction using local anesthesia is impressive. This article traces the history and development of liposuction by dermatologists in the United States. J Drugs Dermatol. 2022;21(9):997-1000. doi:10.36849/JDD.6952.

Randomised, phase 1, dose-finding study of MEDI4166, a PCSK9 antibody and GLP-1 analogue fusion molecule, in overweight or obese patients with type 2 diabetes mellitus.

AIMS/HYPOTHESIS: Cardiovascular disease is the leading cause of morbidity and mortality in people with type 2 diabetes. MEDI4166 is a proprotein convertase subtilisin/kexin type 9 (PCSK9) antibody and glucagon-like peptide-1 (GLP-1) analogue fusion molecule designed to treat patients with type 2 diabetes who are at risk for cardiovascular disease. In this completed, first-in-human study, we evaluated the safety and efficacy of single or multiple doses of MEDI4166 in participants with type 2 diabetes. METHODS: In this phase 1 study that was conducted across 11 clinics in the USA, eligible adults had type 2 diabetes, a BMI of ≥25 kg/m2 to ≤42 kg/m2, and LDL-cholesterol levels ≥1.81 mmol/l. Participants were randomised 3:1 to receive MEDI4166 or placebo using an interactive voice/web response system, which blinded all participants, investigators and study site personnel to the study drug administered. In 'Part A' of the study, five cohorts of participants received a single s.c. injection of MEDI4166 at 10 mg, 30 mg, 100 mg, 200 mg or 400 mg, or placebo. 'Part B' of the study consisted of three cohorts of participants who received an s.c. dose of MEDI4166 once weekly for 5 weeks at 50 mg, 200 mg or 400 mg, or placebo. The primary endpoint in Part A was safety. The co-primary endpoints in Part B were change in LDL-cholesterol levels and area under the plasma glucose concentration-time curve (AUC0-4h) post-mixed-meal tolerance test (MMTT) from baseline to day 36. The pharmacokinetics and immunogenicity of MEDI4166 were also evaluated. RESULTS: MEDI4166 or placebo was administered to n = 30 or n = 10 participants, respectively, in Part A of the study, and n = 48 or n = 15 participants, respectively, in Part B. The incidence of treatment-emergent adverse events (TEAEs) were comparable between MEDI4166 and placebo in both Part A (60% vs 50%) and Part B (79% vs 87%) of the study. Common TEAEs with MEDI4166 included injection-site reactions, diarrhoea and headache; there was no evidence for dose-related increases in TEAEs. In Part B of the study, at all tested doses of MEDI4166, there was a significant decrease in LDL-cholesterol levels vs placebo (least squares mean [95% CI]; MEDI4166 50 mg, -1.25 [-1.66, -0.84]; MEDI4166 200 mg, -1.97 [-2.26, -1.68]; MEDI4166 400 mg, -1.96 [-2.23, -1.70]; placebo, -0.03 [-0.35, 0.28]; all p < 0.0001). However, there were no clinically relevant reductions or significant differences between MEDI4166 vs placebo in glucose AUC0-4h post-MMTT (least squares mean [95% CI]; MEDI4166 50 mg, -10.86 [-17.69, -4.02]; MEDI4166 200 mg, -4.23 [-8.73, 0.28]; MEDI4166 400 mg, -2.59 [-7.14, 1.95]; placebo, -4.84 [-9.95, 0.28]; all p > 0.05). MEDI4166 was associated with a pharmacokinetic profile supportive of weekly dosing and an overall treatment-induced anti-drug antibody-positive rate of 22%. CONCLUSIONS/INTERPRETATION: MEDI4166 was associated with an acceptable tolerability profile and significantly decreased LDL-cholesterol levels in a dose-dependent manner in overweight or obese patients with type 2 diabetes. However, there were no significant reductions in postprandial glucose levels at any dose of MEDI4166. TRIAL REGISTRATION: ClinicalTrials.gov NCT02524782 FUNDING: This study was funded by MedImmune LLC, Gaithersburg, MD, USA.

RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression.

The half-a-tetratricopeptide repeat (HAT) motif is a helical repeat motif found in proteins that influence various aspects of RNA metabolism, including rRNA biogenesis, RNA splicing, and polyadenylation. This functional association with RNA suggested that HAT repeat tracts might bind RNA. However, RNA binding activity has not been reported for any HAT repeat tract, and recent literature has emphasized a protein binding role. In this study, we show that a chloroplast-localized HAT protein, HCF107, is a sequence-specific RNA binding protein. HCF107 consists of 11 tandem HAT repeats and short flanking regions that are also predicted to form helical hairpins. The minimal HCF107 binding site spans ∼11 nt, consistent with the possibility that HAT repeats bind RNA through a modular one repeat-1 nt mechanism. Binding of HCF107 to its native RNA ligand in the psbH 5' UTR remodels local RNA structure and protects the adjacent RNA from exonucleases in vitro. These activities can account for the RNA stabilizing, RNA processing, and translational activation functions attributed to HCF107 based on genetic data. We suggest that analogous activities contribute to the functions of HAT domains found in ribonucleoprotein complexes in the nuclear-cytosolic compartment.

Postprandial dynamics of plasma glucose, insulin, and glucagon in patients with type 2 diabetes treated with saxagliptin plus dapagliflozin add-on to metformin therapy.

OBJECTIVE: To analyze changes in plasma glucose, insulin, and glucagon in relation to glycemic response during treatment with dual add-on of saxagliptin (SAXA) plus dapagliflozin (DAPA) to metformin XR (MET) compared with SAXA add-on or DAPA add-on alone to MET in patients with type 2 diabetes mellitus (T2DM) poorly controlled with MET. METHODS: Double-blind trial in adults with glycated hemoglobin (HbA1c) ≥8.0 to ≤12.0% randomized to SAXA 5 mg/day plus DAPA 10 mg/day (n = 179), or SAXA 5 mg/day and placebo (n = 176), or DAPA 10 mg/day and placebo (n = 179) added to background MET ≥1,500 mg/day. The mean change from baseline in the area under the curve from 0 to 180 minutes (AUC0-180 min) was calculated for glucose, insulin, and glucagon obtained during a liquid meal tolerance test (MTT). RESULTS: Glucose AUC0-180 min was reduced more from baseline with SAXA + DAPA + MET (-12,940 mg/dL) compared with SAXA + MET (-6,309 mg/dL) and DAPA + MET (-11,247 mg/dL). Insulin AUC0-180 min significantly decreased with SAXA + DAPA + MET (-1,120 µU/mL) and DAPA + MET (-1,019 µU/mL) and increased with SAXA + MET (661 µU/mL). Glucagon AUC0-180 min only increased with DAPA + MET (2,346 pg/mL). The changes in glucose (P<.0001) and insulin (P = .0003) AUC0-180 min correlated with change in HbA1c, whereas the change in glucagon AUC0-180 min did not (P = .27). CONCLUSIONS: When added to background MET, the combination of SAXA + DAPA provided additional reductions in glucose AUC0-180 min and HbA1c without the increase in insulin seen with SAXA and without the increase in glucagon seen with DAPA. Changes in insulin and glucose but not glucagon AUC0-180 min correlated with change in HbA1c.

Burden of Uncontrolled Severe Asthma With and Without Elevated Type-2 Inflammatory Biomarkers.

BACKGROUND: Many patients with asthma have type-2 airway inflammation, identified by the presence of biomarkers, including history of allergy, high blood eosinophil (EOS) count, and high fractional exhaled nitric oxide levels. OBJECTIVE: To assess disease burden in relation to type-2 inflammatory biomarker status (history of allergy, blood EOS count, and fractional exhaled nitric oxide level) in patients with uncontrolled and controlled severe asthma in the NOVEL observational longiTudinal studY (NOVELTY) (NCT02760329). METHODS: Asthma diagnosis and severity were physician-reported. Control was defined using Asthma Control Test score (uncontrolled <20, controlled ≥20) and/or 1 or more severe physician-reported exacerbation in the previous year. Biomarker distribution (history of allergy, blood EOS count, and fractional exhaled nitric oxide level), symptom burden (Asthma Control Test score, modified Medical Research Council dyspnea scale), health status (St George's Respiratory Questionnaire score), exacerbations, and health care resource utilization were assessed. RESULTS: Of 647 patients with severe asthma, 446 had uncontrolled and 123 had controlled asthma. Among those with uncontrolled asthma, 196 (44%) had 2 or more positive biomarkers, 187 (42%) had 1 positive biomarker, 325 (73%) had low blood EOS, and 63 (14%) were triple-negative. Disease burden was similarly high across uncontrolled subgroups, irrespective of biomarker status, with poor symptom control (Asthma Control Test score 14.9-16.6), impaired health status (St George's Respiratory Questionnaire total score 46.7-49.4), clinically important breathlessness (modified Medical Research Council grade ≥2 in 47.3%-57.1%), and 1 or more severe exacerbation (70.6%-76.2%). CONCLUSIONS: Type-2 inflammatory biomarkers did not differentiate disease burden in patients with severe asthma. Patients with low type-2 inflammatory biomarker levels have few biologic therapy options; their needs should be addressed.

Symptom Burden, Health Status, and Productivity in Patients with Uncontrolled and Controlled Severe Asthma in NOVELTY.

Background: Few studies have quantified symptom burden, health status, and productivity in patients with uncontrolled and controlled severe asthma. Up-to-date, real-world, global evidence is needed. Objective: To quantify symptom burden, health status, and productivity in patients with uncontrolled and controlled severe asthma using baseline data from the NOVEL observational longiTudinal studY (NOVELTY; NCT02760329). Methods: NOVELTY included patients aged ≥18 years (or ≥12 years in some countries) from primary care and specialist centres in 19 countries, with a physician-assigned diagnosis of asthma, asthma+chronic obstructive pulmonary disease (COPD), or COPD. Disease severity was physician-assessed. Uncontrolled severe asthma was defined by an Asthma Control Test (ACT) score <20 and/or severe physician-reported exacerbations in the previous year; controlled severe asthma required an ACT score ≥20 and no severe exacerbations. Assessment of symptom burden included Respiratory Symptoms Questionnaire (RSQ) and ACT score. Assessment of health status included St George's Respiratory Questionnaire (SGRQ), EuroQoL 5 Dimensions 5 Levels Health Questionnaire (EQ-5D-5L) index value, and EQ-5D-5L Visual Analog Score (EQ-VAS). Assessment of productivity loss included absenteeism, presenteeism, overall work impairment, and activity impairment. Results: Of 1652 patients with severe asthma, asthma was uncontrolled in 1078 (65.3%; mean age 52.6 years, 65.8% female) and controlled in 315 (19.1%; mean age 55.2 years, 56.5% female). With uncontrolled versus controlled severe asthma, symptom burden was higher (mean RSQ score 7.7 vs 2.5), health status more impaired (mean SGRQ total score 47.5 vs 22.4; mean EQ-5D-5L index value 0.68 vs 0.90; mean EQ-VAS score 64.1 vs 78.1), and productivity lower (presenteeism 29.3% vs 10.5%). Conclusion: Our findings highlight the symptom burden of uncontrolled severe asthma compared with controlled severe asthma and its impact on patient health status and productivity, and support the need for interventions to improve control of severe asthma.

Iron requirements based upon iron absorption tests are poorly predicted by haematological indices in patients with inactive inflammatory bowel disease.

Fe deficiency and Fe-deficiency anaemia are common in patients with inflammatory bowel disease (IBD). Traditional clinical markers of Fe status can be skewed in the presence of inflammation, meaning that a patient's Fe status can be misinterpreted. Additionally, Fe absorption is known to be down-regulated in patients with active IBD. However, whether this is the case for quiescent or mildly active disease has not been formally assessed. The present study aimed to investigate the relationship between Fe absorption, Fe requirements and standard haematological indices in IBD patients without active disease. A group of twenty-nine patients with quiescent or mildly active IBD and twenty-eight control subjects undertook an Fe absorption test that measured sequential rises in serum Fe over 4 h following ingestion of 200 mg ferrous sulphate. At baseline, serum Fe, transferrin saturation, non-transferrin-bound Fe (NTBI), ferritin and soluble transferrin receptor were all measured. Thereafter (30-240 min), only serum Fe and NTBI were measured. Fe absorption did not differ between the two groups (P = 0·9; repeated-measures ANOVA). In control subjects, baseline haematological parameters predicted Fe absorption (i.e. Fe requirements), but this was not the case for patients with IBD. Fe absorption is normal in quiescent or mildly active IBD patients but standard haematological parameters do not accurately predict Fe requirements.

Structure of the catalytic domain of Plasmodium falciparum ARF GTPase-activating protein (ARFGAP).

The crystal structure of the catalytic domain of the ADP ribosylation factor GTPase-activating protein (ARFGAP) from Plasmodium falciparum has been determined and refined to 2.4 Å resolution. Multiwavength anomalous diffraction (MAD) data were collected utilizing the Zn(2+) ion bound at the zinc-finger domain and were used to solve the structure. The overall structure of the domain is similar to those of mammalian ARFGAPs. However, several amino-acid residues in the area where GAP interacts with ARF1 differ in P. falciparum ARFGAP. Moreover, a number of residues that form the dimer interface in the crystal structure are unique in P. falciparum ARFGAP.

Comparative Analysis of Lumbar Spine Vertebral Morphology Between Maori and New Zealand Europeans: A Computed Tomography Study.

BACKGROUND: Most existing studies of lumbar anatomy do not consider ethnic influence and recruit mostly white participants. Recent studies have considered other populations; however, none have assessed Maori, the indigenous people of New Zealand (NZ). A computed tomography study of vertebral body (VB) and canal dimensions was performed for lumbar vertebrae of Maori and NZ European patients to evaluate for ethnic variation. METHODS: Lumbar vertebrae from 196 patients were measured using computed tomography. After interrater and intrarater reliability analyses, a single trained examiner measured VB heights, VB lengths, segmental angle, pedicle height and width, and vertebral canal length (VCL) and vertebral canal width for each level. Canal:body ratio was calculated. Demographic data recorded included age, sex, and ethnicity. RESULTS: VCL remained relatively constant through the lumbar spine; canal width increased to a maximum of 28.2 mm at L5. Canal:body ratios and pedicle height decreased while pedicle width increased to a maximum of 16.1 mm at L5. There were few differences between Maori and NZ Europeans except at the L5 level, where VCL and canal:body ratio were larger in NZ Europeans (P < 0.05), and pedicle height, width, and VB pediculolaminar length were larger in Maori (P < 0.05). Females had generally smaller measurements and age was a positive predictor of measured values (P < 0.05). CONCLUSIONS: This study is the first to characterize lumbar anatomy in a Maori cohort. Adequately powered results demonstrated few differences between Maori and NZ Europeans. Isolated differences observed at L5 may be due to sacropelvic differences, which represent an area for further investigation. LEVEL OF EVIDENCE: 3. CLINICAL RELEVANCE: Diagnosis, surgical planning, ethnic differences.

Structure of human desArg-C5a.

The anaphylatoxin C5a is derived from the complement component C5 during activation of the complement cascade. It is an important component in the pathogenesis of a number of inflammatory diseases. NMR structures of human and porcine C5a have been reported; these revealed a four-helix bundle stabilized by three disulfide bonds. The crystal structure of human desArg-C5a has now been determined in two crystal forms. Surprisingly, the protein crystallizes as a dimer and each monomer in the dimer has a three-helix core instead of the four-helix bundle noted in the NMR structure determinations. Furthermore, the N-terminal helices of the two monomers occupy different positions relative to the three-helix core and are completely different from the NMR structures. The physiological significance of these structural differences is unknown.

A case of infection-associated antiproteinase-3-negative cytoplasmic antineutrophil cytoplasmic antibody pauci-immune focal necrotizing glomerulonephritis.

We present the case of a man with Gram-negative sepsis and exposure to oral silica who developed pauci-immune focal necrotizing glomerulonephritis (PI-FNGN) in the setting of a subacute polymicrobial central venous line (CVL) infection. He developed a cytoplasmic antineutrophil cytoplasmic autoantibody (C-ANCA) that was antiproteinase-3 (PR-3) and antimyeloperoxidase (MPO) antibody negative. We believe this is the first reported case of Gram-negative sepsis-associated PI-FNGN. Chronic silica exposure is a leading environmental risk factor in the development of ANCA vasculitis. Oral silica is a common pharmaceutical additive and its bioavailability is being recognized. Oral silica, therefore, may also be a risk for development of autoreactivity. The PI-FNGN resolved with antibiotic therapy alone. The C-ANCA titer declined as the PI-FNGN resolved. The case supports experimental and observational research that environmental exposures act as adjuvants for an immune response and also provide epigenetic triggers for autoreactivity. The C-ANCA was negative for PR-3, its major antigen. C-ANCA antigen specificity may depend on the pathogenesis of the underlying disease, potentially elicited by a cross-reaction of an antibody to foreign and self target antigen sequence homology or alternatively elicited by antigenic epitope spread.

Scoring system for renal pathology in Fabry disease: report of the International Study Group of Fabry Nephropathy (ISGFN).

BACKGROUND: In Fabry nephropathy, alpha-galactosidase deficiency leads to accumulation of glycosphingolipids in all kidney cell types, proteinuria and progressive loss of kidney function. METHODS: An international working group of nephrologists from 11 Fabry centres identified adult Fabry patients, and pathologists scored histologic changes on renal biopsies. A standardized scoring system was developed with a modified Delphi technique assessing 59 Fabry nephropathy cases. Each case was scored independently of clinical information by at least three pathologists with an average final score reported. RESULTS: We assessed 35 males (mean age 36.4 years) and 24 females (43.9 years) who mostly had clinically mild Fabry nephropathy. The average serum creatinine was 1.3 mg/dl (114.9 micromol/l); estimated glomerular filtration rate was 81.7 ml/min/1.73 m(2) and urine protein to creatinine ratio was 1.08 g/g (122.0 mg/mmol). Males had greater podocyte vacuolization on light microscopy (mean score) and glycosphingolipid inclusions on semi-thin sections than females. Males also had significantly more proximal tubule, peritubular capillary and vascular intimal inclusions. Arteriolar hyalinosis was similar, but females had significantly more arterial hyalinosis. Chronic kidney disease stage correlated with arterial and glomerular sclerosis scores. Significant changes, including segmental and global sclerosis, and interstitial fibrosis were seen even in patients with stage 1-2 chronic kidney disease with minimal proteinuria. CONCLUSIONS: The development of a standardized scoring system of both disease-specific lesions, i.e. lipid deposition related, and general lesions of progression, i.e. fibrosis and sclerosis, showed a spectrum of histologic appearances even in early clinical stage of Fabry nephropathy. These findings support the role of kidney biopsy in the baseline evaluation of Fabry nephropathy, even with mild clinical disease. The scoring system will be useful for longitudinal assessment of prognosis and responses to therapy for Fabry nephropathy.

Structure of Plasmodium falciparum ADP-ribosylation factor 1.

Vesicular trafficking may play a crucial role in the pathogenesis and survival of the malaria parasite. ADP-ribosylation factors (ARFs) are among the major components of vesicular trafficking pathways in eukaryotes. The crystal structure of ARF1 GTPase from Plasmodium falciparum has been determined in the GDP-bound conformation at 2.5 Šresolution and is compared with the structures of mammalian ARF1s.

Retrospective evaluation of prognostic factors in metastatic spine disease: serum albumin and primary tumour type are key.

BACKGROUND: Treatment decisions for metastatic spine disease are complex and depend on prognosis. Four prognostic systems in use are the Oswestry Risk Index, modified Bauer score (MBS), van der Linden score and New England Spinal Metastasis Score (NESMS). We aimed to determine the performance of these scoring systems in a New Zealand cohort of patients and develop a prognostic score specific to this demographic. METHODS: A retrospective review of a patient cohort from 2009 to 2016 was undertaken. Scores and individual scoring items were evaluated with univariate and multivariate analyses. Significant items were used to design a simple, population-specific objective scoring system, which was then tested. RESULTS: A total of 106 patients receiving either surgery and radiotherapy (65) or radiotherapy alone (41) were included. Mean post-treatment survival time was 13.7 months. All scoring systems were significantly correlated with survival and had similar concordances. The MBS had the largest coefficient of determination (Cox and Snell's R2 = 0.18), followed by the NESMS (R2 = 0.14). On multivariate analysis, the lung cancer (MBS) and serum albumin (NESMS) items were significant. A modified Oswestry Risk Index primary tumour item and NESMS serum albumin outperformed the MBS (R2 = 0.20), providing the basis for a prognostic scoring tool specific to our demographic. CONCLUSION: Based on serum albumin and primary tumour type, we propose the 'Metastatic Spine Risk Index' as a simple and objective tool, specific to our population for predicting survival, which can be used in conjunction with other clinical information when considering treatment options.

AAV Targeting of Glial Cell Types in the Central and Peripheral Nervous System and Relevance to Human Gene Therapy.

Different glial cell types are found throughout the central (CNS) and peripheral nervous system (PNS), where they have important functions. These cell types are also involved in nervous system pathology, playing roles in neurodegenerative disease and following trauma in the brain and spinal cord (astrocytes, microglia, oligodendrocytes), nerve degeneration and development of pain in peripheral nerves (Schwann cells, satellite cells), retinal diseases (Müller glia) and gut dysbiosis (enteric glia). These cell type have all been proposed as potential targets for treating these conditions. One approach to target these cell types is the use of gene therapy to modify gene expression. Adeno-associated virus (AAV) vectors have been shown to be safe and effective in targeting cells in the nervous system and have been used in a number of clinical trials. To date, a number of studies have tested the use of different AAV serotypes and cell-specific promoters to increase glial cell tropism and expression. However, true glial-cell specific targeting for a particular glial cell type remains elusive. This review provides an overview of research into developing glial specific gene therapy and discusses some of the issues that still need to be addressed to make glial cell gene therapy a clinical reality.

An unexpected phosphate binding site in glyceraldehyde 3-phosphate dehydrogenase: crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme.

BACKGROUND: The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. RESULTS: We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2A resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits. CONCLUSION: The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.

Effectiveness of perturbation theory approaches for computing non-condon electron transfer dynamics in condensed phases.

A description of electron transfer in condensed-phase media requires models that adequately describe the coupling of the electronic degrees of freedom to the surrounding nuclear coordinates. The spin-boson model has been the canonical model used to understand quantum dynamic processes in condensed-phase media over the last 25 years. Inherent in the standard model of a two-state quantum system coupled to a bosonic bath is the assumption that the Condon approximation is valid. In this context, the Condon approximation assumes that the bath configurations (coordinates) have no effect on the nonadiabatic coupling matrix element. While this is a useful model for electron transfer in small molecular systems, the validity of this approximation is less likely when large-scale motions of solvent molecules are strongly coupled to the electron transfer event, e.g., in molecular clamps and long-range electron transfer in biopolymers. In the present paper a general model for two-state electron transfer which allows for system-bath coupling in both the diagonal and off-diagonal (nonadiabatic) terms is studied. Time-dependent perturbation theory for this Hamiltonian is developed using a small polaron transformation. As noted in several recent studies, in a certain regime of parameter space, the relevant Hamiltonian admits an exact solution, termed the exactly solvable non-Condon Hamiltonian (or NCE). This limit, for which exact solutions are available, is used to benchmark the short- and long-time accuracy of various perturbative approaches. The validated perturbation equations are subsequently used to explore the role of non-Condon effects on electron transfer by systematically increasing the strength of the non-Condon coupling term from zero (i.e., the canonical spin-boson model) to the value that pertains to the exactly solvable non-Condon model (where non-Condon effects are significant).

Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling.

We and others have demonstrated that Fas-mediated apoptosis is a potential therapeutic target for cholangiocarcinoma. Previously, we reported that CaM (calmodulin) antagonists induced apoptosis in cholangiocarcinoma cells through Fas-related mechanisms. Further, we identified a direct interaction between CaM and Fas with recruitment of CaM into the Fas-mediated DISC (death-inducing signalling complex), suggesting a novel role for CaM in Fas signalling. Therefore we characterized the interaction of CaM with proteins recruited into the Fas-mediated DISC, including FADD (Fas-associated death domain)-containing protein, caspase 8 and c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1beta-converting enzyme]-like inhibitory protein}. A Ca(2+)-dependent direct interaction between CaM and FLIP(L), but not FADD or caspase 8, was demonstrated. Furthermore, a 37.3+/-5.7% increase (n=6, P=0.001) in CaM-FLIP binding was observed at 30 min after Fas stimulation, which returned to the baseline after 60 min and correlated with a Fas-induced increase in intracellular Ca(2+) that reached a peak at 30 min and decreased gradually over 60 min in cholangiocarcinoma cells. A CaM antagonist, TFP (trifluoperazine), inhibited the Fas-induced increase in CaM-FLIP binding concurrent with inhibition of ERK (extracellular-signal-regulated kinase) phosphorylation, a downstream signal of FLIP. Direct binding between CaM and FLIP(L) was demonstrated using recombinant proteins, and a CaM-binding region was identified in amino acids 197-213 of FLIP(L). Compared with overexpression of wild-type FLIP(L) that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIP(L) with deletion of the CaM-binding region resulted in increased spontaneous and Fas-induced apoptosis in cholangiocarcinoma cells. Understanding the biology of CaM-FLIP binding may provide new therapeutic targets for cholangiocarcinoma and possibly other cancers.