Comparison of a Novel Score "NOD-ACLF" to Other Established Prognostic Scores for Prediction of Mortality in APASL-ACLF Patients: A Cohort Study from a Tertiary Care Center of North India.

Background: Commonly used prognostic scores for acute on-chronic liver failure (ACLF) have complex calculations. We tried to compare the simple counting of numbers and types of organ dysfunction to these scores, to predict mortality in ACLF patients. Methods: In this prospective cohort study, ACLF patients diagnosed on the basis of Asia Pacific Association for Study of the Liver (APASL) definition were included. Severity scores were calculated. Prognostic factors for outcome were analysed. A new score, the Number of Organ Dysfunctions in Acute-on-Chronic Liver Failure (NOD-ACLF) score was developed. Results: Among 80 ACLF patients, 74 (92.5%) were male, and 6 were female (7.5%). The mean age was 41.0±10.7 (18-70) years. Profile of acute insult was; alcohol 48 (60%), sepsis 30 (37.5%), variceal bleeding 22 (27.5%), viral 8 (10%), and drug-induced 3 (3.8%). Profiles of chronic insults were alcohol 61 (76.3%), viral 20 (25%), autoimmune 3 (3.8%), and non-alcoholic steatohepatitis 2 (2.5%). Thirty-eight (47.5%) were discharged, and 42 (52.5%) expired. The mean number of organ dysfunction (NOD-ACLF score) was ->4.5, simple organ failure count (SOFC) score was >2.5, APASL ACLF Research Consortium score was >11.5, Model for End-Stage Liver Disease-Lactate (MELD-LA) score was >21.5, and presence of cardiovascular and respiratory dysfunctions were significantly associated with mortality. NOD-ACLF and SOFC scores had the highest area under the receiver operating characteristic to predict mortality among all these. Conclusion: The NOD-ACLF score is easy to calculate bedside and is a good predictor of mortality in ACLF patients performing similar or better to other scores.

KGHeBTA (King George's Medical University Hepatitis B Therapeutic Algorithm): A New Diagnostic and Therapeutic Algorithm and Clinico-epidemiological Spectrum of Hepatitis B.

Background: Diagnostic and therapeutic algorithms given by various societies for hepatitis B are fragmented and complex. The clinico-epidemiologic spectrum of hepatitis B is not studied with large-scale data from our region. We aimed to develop a comprehensive algorithm for the treatment of hepatitis B and study its clinico-epidemiological spectrum. Methods: From 2014-2019, the clinico-laboratory data of hepatitis B surface antigen (HbsAg)-positive patients were prospectively recorded. King George's Medical University hepatitis B therapeutic algorithm (KGHeBTA) was developed on the basis of the standard existing guidelines. The prevalence of different clinical stages of HBsAg-positive patients was calculated and their treatment records reviewed. Testing circumstances and risk factors were noted. Results: Among 1,508 data record sheets, 421 were complete. According to the KGHeBTA algorithm, 221 had detectable hepatitis B virus DNA. 21% were cirrhotic and 79% non-cirrhotic. 72% were incidentally detected asymptomatic hepatitis B, 7% were hepatitis B with acute symptoms, 0.7% were acute hepatitis B, and 22% were chronic hepatitis B. 20% patients were eligible for antivirals and 80% patients were not eligible. 32% patients were actually treated with antivirals due to the inclusion of some special indications as pregnancy and family history. Screening during various medical illnesses (40%) was the most common and during health camps (0.2%), the least common testing approach. Road-side shaving (52%) was the most common and intravenous drug abuse (0.2%) and the least common risk factor for the detection of hepatitis B in our data pool. Conclusions: HBsAg-positive patients can be easily worked up and treated based on the proposed algorithm (KGHeBTA). About one fourth to one fifth of all HBsAg-positive patients were eligible and treated with oral antivirals. Most of the patients were incidentally detected asymptomatic hepatitis B screened during medical illnesses. Roadside shaving and intravenous drug abuse were the most and the least common risk factors.

Obstructive Sleep Apnea in Hypertension.

Introduction About one-half of patients who have essential hypertension have obstructive sleep apnea (OSA), and about one-half of patients who have obstructive sleep apnea have essential hypertension. OSA can cause even resistant hypertension if left untreated. These two entities often co-exist and are seen as a continuum of the same process. Eighty percent to 90% of OSA cases are undiagnosed mostly because of a lack of awareness. Material and methods This was a cross-sectional study done over a period of one year in a tertiary care hospital. After taking informed consent, 179 hypertensive patients of >18 years were included in the study. All patients were screened for OSA by the STOP-BANG questionnaire. Patients having scores of ≥3 were subjected to overnight polysomnography to confirm the diagnosis of OSA (AHI ≥5). Patients with a STOP-BANG score ≤2 or score ≥3 with AHI <5, were considered non-OSA. Results More than half (53.1%) of the patients enrolled in the study had OSA. Their age ranged from 18 to 78 years and the mean age was 52.07±11.40 years. The mean age of OSA cases was found to be slightly higher than that of non-OSA. The majority of OSA cases were males (73.7%). There was an increase in the prevalence, as well as the severity of OSA, with an increase in BMI. Most of the cases had snoring and a history of tiredness. Triglyceride (TG) and low-density lipoprotein (LDL) levels of the OSA group were found to be significantly higher and high-density lipoprotein (HDL) levels were significantly lower than that of the non-OSA group. Conclusion More than half of our hypertensive patients had OSA. These two conditions often co-exist and are known as a dangerous pair. Physicians ought to have increased suspicion for early diagnosis and treatment to improve cardiovascular outcomes, reduce road traffic accidents, and improve quality of life.

Amphotericin B induced pancytopenia.

Amphotericin B (AMB) has been the irreplaceable drug of choice for countless fungal and protozoal infections. One of the lesser-known adverse effects of AMB is Pancytopenia - very rare with very few cases reported - most commonly observed following prolonged administration. We report the case of a patient suffering from visceral leishmaniasis, who developed worsening pancytopenia four to five days after being administered a single bolus dose of Liposomal Amphotericin B (L-AMB). The diagnosis was clinical and management involved supportive care, and granulocyte-macrophage colony-stimulating factor (GM-CSF). AMB is an effective drug, but is also associated with numerous side effects. Physicians are well-versed with the more frequently seen adverse drug reactions and their management. However, pancytopenia, being a rare adverse reaction to AMB, is less known and can be easily overlooked. This case report aims to ensure that the physicians must be aware of such possibilities in the first place to make swift diagnoses and management. The condition itself is seemingly self-limiting, although GM-CSF may be needed in refractory cases. It's true that few previous case reports have indicated pancytopenia in association with prolonged AMB exposure, but we believe certain conditions may predispose a patient to a more acute presentation - as seen in our case.

Prevalence of obstructive sleep apnea in diabetic patients.

Context: Obstructive sleep apnea (OSA)-related hypoxemia stimulates release of acute-phase proteins and reactive oxygen species that exacerbate insulin resistance and lipolysis and cause an augmented prothrombotic and proinflammatory state which can leads to premature death. Aims: This study aims to study the prevalence of OSA in diabetic patients. Setting and Design: It was a cross-sectional study, done over a period of 1 year in a tertiary care hospital. Materials and Methods: A total of 149 type 2 diabetic patients were enrolled after taking written consent. All patients were subjected to STOP BANG questionnaire and patients falling in intermediate-high risk (score 3-8), were taken for overnight polysomnography to confirm the diagnosis of OSA (apnea hypopnea index ≥ 5). Statistical Analysis Used: Statistical Package for Social Sciences (SPSS) Version 21.0 statistical analysis software. Results: Fifty-five percent of our diabetic population were having OSA. The age of patients enrolled in the study ranged between 30 and 86 years and prevalence increases with an increase in age groups. Majority (61.7%) of our cases were males. Incremental trend in weight, body mass index (BMI), neck circumference, and waist circumference of OSA cases were found with increasing in severity of OSA. Mean levels of raised blood sugar and HbA1c were higher in severe OSA cases. Conclusions: OSA has a high prevalence in patients with type 2 diabetes mellitus. Patients with type 2 diabetes should be screened for OSA, even in the absence of symptoms, especially in individuals with higher waist circumference and BMI.

RésuméContexte: L'hypoxémie liée à l'apnée obstructive du sommeil (AOS) stimule la libération de protéines de phase aiguë et d'espèces réactives de l'oxygène qui exacerber la résistance à l'insuline et la lipolyse et provoquer un état prothrombotique et pro-inflammatoire accru qui peut conduire à un décès. Objectifs: Cette étude vise à étudier la prévalence de l'AOS chez les patients diabétiques. Cadre et conception: il s'agissait d'une étude transversale, réalisée sur une période d'un an dans un hôpital de soins tertiaires. Matériels et méthodes: Un total de 149 patients diabétiques de type 2 ont été recrutés après avoir pris consentement écrit. Tous les patients ont été soumis au questionnaire STOP BANG et les patients à risque intermédiaire-élevé (score 3-8), ont été prise pour une polysomnographie nocturne pour confirmer le diagnostic d'AOS (index d'apnée hypopnée ≥ 5). Analyse statistique utilisée: statistique Package for Social Sciences (SPSS) Version 21.0 du logiciel d'analyse statistique. Résultats: Cinquante-cinq pour cent de notre population diabétique étaient souffrant d'AOS. L'âge des patients inclus dans l'étude variait entre 30 et 86 ans et la prévalence augmente avec l'augmentation des tranches d'âge. La majorité (61,7 %) de nos cas étaient des hommes. Tendance progressive du poids, de l'indice de masse corporelle (IMC), du tour de cou et du tour de taille des cas d'AOS ont été trouvés avec une augmentation de la sévérité de l'AOS. Les taux moyens de glycémie élevée et d'HbA1c étaient plus élevés dans les cas graves d'AOS. Conclusions: L'AOS a une prévalence élevée chez les patients atteints de diabète de type 2. Les patients atteints de diabète de type 2 doivent subir un dépistage de l'AOS, même en l'absence de symptômes, en particulier chez les personnes ayant un tour de taille et un IMC plus élevés.

Clinical and biochemical profile of scrub typhus patients at a tertiary care hospital in Northern India.

BACKGROUND: Scrub typhus is a neglected rickettsial disease in India. Every year, we are facing outbreaks of Scrub typhus after Monsoon season. Patients present with a wide clinical spectrum ranging from pyrexia of unknown origin to multiple organ dysfunction. Some of these clinical features overlap with presentation of other tropical infections prevalent in Indian subcontinent, which leads to diagnostic dilemma and delay in diagnosis. Hence, we planned this study to know the demographic, clinical and biochemical profile of scrub typhus patients. METHODS: This was an observational study conducted in department of Medicine, King George's Medical University Lucknow, India a leading tertiary care hospital of Northern India. All scrub typhus patients were evaluated by detailed history, examination and laboratory tests. RESULTS: We enrolled 52 patients in our study. The mean age of the patients was 35.17 ± 16.90 years with majority (82.7%) of patients from rural background. All the patients had fever with an average duration of 9.6 ± 2 days. Most of the patients developed hepatitis (69.2%) followed by acute encephalitis syndrome (47%), acute kidney injury (23.1%) and acute respiratory failure (19.2%). Eschar was found in 11 patients (21.2%). CONCLUSION: Scrub typhus is often misdiagnosed or diagnosed late due to its wide clinical spectrum overlapping with clinical presentation of other commonly prevalent tropical diseases. One should always consider the differential diagnosis of scrub typhus while evaluating a young febrile patient of rural background, with features of single or multiple organ dysfunction and laboratory findings of leucocytosis, thrombocytopenia and elevation of transaminases.

Adhesion molecules, chemokines and matrix metallo-proteinases response after albendazole and albendazole plus steroid therapy in swine neurocysticercosis.

The treatment of neurocysticercosis (NCC) varies with location, number and stage of the Taenia solium cysticerci (cysts). Albendazole (ABZ) effectively kills cysticerci, and subsequently induces neuro-inflammation facilitated by leukocyte infiltration. We hypothesize that immune response varies around drug responder (degenerating/dying) and non-responder (viable) cysts after ABZ and ABZ plus steroid (ABZS) therapy, which may determine the disease pathogenesis. Twenty cysticercotic swine were treated with ABZ (n = 10; group1) and ABZS (n = 10; group2). Expression of adhesion molecules, chemokines and matrix metallo-proteinases (MMPs) was measured by qRT-PCR (quantitative reverse transcriptase-polymerase chain reaction) and ELISA. Gelatin gel zymography was performed to detect the activity of MMP-2 and -9. In group1, ABZ therapy induced higher expressions of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), E-selectin, MCP-1 (monocyte chemotactic protein-1), Eotaxin-1, MIP-1α (macrophage inflammatory protein-1α), RANTES (regulated on activation, normal T cell expressed and secreted), MMP-2 and MMP-9 around ABZ responder (AR) cysts. Three pigs with cyst burdens ≥10 died following ABZ therapy. However, in group2, moderate expressions of ICAM-1, VCAM-1, E-selectin, RANTES and MMP-9 were associated with ABZS responder (ASR), whereas low expressions of these molecules were associated with ABZS non-responder (ASNR) cysts. In conclusion, ABZ alone therapy is not safe since it causes death of pigs due to higher inflammatory immune response around dying cysts. However, combination therapy is an effective treatment regimen even with the high cyst burden.

Plasminogen Activator Inhibitor-1 Protects Mice Against Cardiac Fibrosis by Inhibiting Urokinase-type Plasminogen Activator-mediated Plasminogen Activation.

Plasminogen activator inhibitor-1 (PAI-1) is known to protect mice against cardiac fibrosis. It has been speculated that PAI-1 may regulate cardiac fibrosis by inactivating urokinase-type plasminogen activator (uPA) and ultimately plasmin (Pm) generation. However, the in vivo role of PAI-1 in inactivating uPA and limiting the generation of Pm during cardiac fibrosis remains to be established. The objective of this study was to determine if the cardioprotective effect of PAI-1 is mediated through its ability to directly regulate urokinase -mediated activation of plasminogen (Pg). An Angiotensin II (AngII)-aldosterone (Ald) infusion mouse model of hypertension was utilised in this study. Four weeks after AngII-Ald infusion, PAI-1-deficient (PAI-1-/-) mice developed severe cardiac fibrosis. However, a marked reduction in cardiac fibrosis was observed in PAI-1-/-/uPA-/- double knockout mice that was associated with reduced inflammation, lower expression levels of TGF-ß and proteases associated with tissue remodeling, and diminished Smad2 signaling. Moreover, total ablation of cardiac fibrosis was observed in PAI-1-/- mice that express inactive plasmin (Pm) but normal levels of zymogen Pg (PAI-1-/-/PgS743A/S743A). Our findings indicate that PAI-1 protects mice from hypertension-induced cardiac fibrosis by inhibiting the generation of active Pm.

Human Glutathione S-Transferase Enzyme Gene Polymorphisms and Their Association With Neurocysticercosis.

Neurocysticercosis (NCC), caused by cysticerci of Taenia solium is the most common helminthic infection of the central nervous system. Some individuals harboring different stages of cysticerci in the brain remain asymptomatic, while others with similar cysticerci lesions develop symptoms and the reasons remain largely unknown. Inflammatory response to antigens of dying parasite is said to be responsible for symptomatic disease. Reactive oxygen species (ROS) that are generated in inflammatory conditions can damage cellular macromolecules such as lipids, DNA, and proteins. The glutathione S-transferases (GSTs) are critical for the protection of cells from ROS. A total of 250 individuals were included in the study: symptomatic NCC = 75, asymptomatic NCC = 75, and healthy controls = 100. The individuals carrying the deletions of GSTM1 and GSTT1 were at risk for NCC (OR = 2.99, 95 %CI = 1.31-6.82, p = 0.0073 and OR = 1.94, 95 %CI = 0.98-3.82, p = 0.0550 respectively). Further, the individuals with these deletions were more likely to develop symptomatic disease (OR = 5.08, 95 % CI = 2.12-12.18, p = 0.0001 for GSTM1 and OR = 3.25, 95 %CI = 1.55-6.82, p = 0.0018 for GSTT1). Genetic variants of GSTM3 and GSTP1 were not associated with NCC. The total GST activity and levels of GSTM1, GSTT1, and GSTM3 were significantly higher in asymptomatic subjects than in symptomatic and healthy controls. Lower GST activity was observed in individuals with GSTM1 and GSTT1 deletions. The present study suggests that the individuals with GSTM1 and GSTT1 deletions are at higher risk to develop symptomatic disease. The higher GST activity and levels of GSTM1, GSTT1, and GSTM3 are likely to play role in maintaining asymptomatic condition.

Identification of species and genetic variation in Taenia isolates from human and swine of North India.

Taenia solium is the major cause of taeniasis and cysticercosis/neurocysticercosis (NCC) in the developing countries including India, but the existence of other Taenia species and genetic variation have not been studied in India. So, we studied the existence of different Taenia species, and sequence variation in Taenia isolates from human (proglottids and cysticerci) and swine (cysticerci) in North India. Amplification of cytochrome c oxidase subunit 1 gene (cox1) was done by polymerase chain reaction (PCR) followed by sequencing and phylogenetic analysis. We identified two species of Taenia i.e. T. solium and Taenia asiatica in our isolates. T. solium isolates showed similarity with Asian genotype and nucleotide variations from 0.25 to 1.01 %, whereas T. asiatica displayed nucleotide variations ranged from 0.25 to 0.5 %. These findings displayed the minimal genetic variations in North Indian isolates of T. solium and T. asiatica.

Plasminogen activator inhibitor-1 stimulates macrophage activation through Toll-like Receptor-4.

While inflammation is often associated with increased Plasminogen Activator Inhibitor-1 (PAI-1), the functional consequences of PAI-1 in inflammation have yet to be fully determined. The aim of this study was to establish the in vivo relevance of PAI-1 in inflammation. A mouse model of systemic inflammation was employed in wild-type (WT) and PAI-1 deficient (PAI-1(-/-)) mice. Mice survival, macrophage infiltration into the lungs, and plasma levels of pro-inflammatory cytokines were assessed after lipopolysaccharide (LPS) infusion. In vitro experiments were conducted to examine changes in LPS-induced inflammatory responses after PAI-1 exposure. PAI-1 was shown to regulate inflammation, in vivo, and affect macrophage infiltration into lungs. Further, PAI-1 activated macrophages, and increased pro-inflammatory cytokines at both the mRNA and protein levels in these cells. The effect of PAI-1 on macrophage activation was dose-dependent and LPS-independent. Proteolytic inhibitory activity and Lipoprotein Receptor-related Protein (LRP) and vitronectin (VN) binding functions, were not involved in PAI-1-mediated activation of macrophages. However, the effect of PAI-1 on macrophage activation was partially blocked by a TLR4 neutralizing antibody. Furthermore, PAI-1-induced Tumor Necrosis Factor-alpha (TNF-α) and Macrophage Inflammatory Protein-2 (MIP-2) expression was reduced in TLR4(-/-) macrophages compared to WT macrophages. These results demonstrate that PAI-1 is involved in the regulation of host inflammatory responses through Toll-like Receptor-4 (TLR4)-mediated macrophage activation.

Drug reaction with eosinophilia and systemic symptoms syndrome associated with Nitrofurantoin.

Drug reaction with eosinophilia and systemic symptom (DRESS) is a severe adverse drug-induced reaction with a prolonged latency period which is characterized by a variety of clinical manifestations, usually fever, rash, lymphadenopathy, eosinophilia, and a wide range of mild-to-severe systemic presentations. Drugs are an important cause of DRESS in most of the cases. It is challenging to diagnose DRESS because of the diversity of cutaneous eruption and visceral organs involvement. We hereby report a 34-year-old female who developed DRESS syndrome following ingestion of nitrofurantoin for the treatment of urinary tract infection. She was managed conservatively and recovered after few weeks. Our aim of this study is to raise awareness to suspect DRESS syndrome in patients who present with unusual clinical features with skin involvement after initiating any drug.

Immune response to Taenia solium cysticerci after anti-parasitic therapy.

Albendazole is the drug of choice for Taenia solium infection. Concomitant administration of steroid has been advocated to avoid adverse reactions to albendazole therapy in neurocysticercosis. Some T. solium cysticerci (larvae) respond to albendazole therapy while others do not and the reasons remain unexplained. We hypothesise that the immune response differs between treatment responder and non-responder cysticerci and this may determine the outcome. Twenty swine naturally infected with T. solium were purchased from the market and the infection was confirmed by magnetic resonance imaging. Swine were divided into two groups; swine in group 1 were treated with albendazole and those in group 2 were treated with albendazole plus steroid (prednisolone). All the animals underwent follow-up MRIs at 6 and 12 weeks after start of therapy and were then sacrificed. Tissues surrounding the cysticerci were collected and studied for the expression of different cytokines by reverse transcriptase PCR and ELISA. Albendazole therapy was found to be more effective in parasite killing than albendazole plus steroid (94.11% versus 70.96%, P=0.011). Albendazole therapy provoked a pro-inflammatory, Th1 (IFN-γ) and pleiotropic (IL-6) cytokine response around the dead cysticerci. Despite a heavy parasite burden in the brain, all the pigs treated with albendazole plus steroid survived. In this group of animals, a mixed pro-inflammatory Th1, Th2 (IL-4) and regulatory cytokine (IL-10) response was associated with responder cysticerci. Further, Th2 and regulatory cytokine responses were associated with non-responder cysticerci.

Design, synthesis and biological evaluation of di-substituted noscapine analogs as potent and microtubule-targeted anticancer agents.

Noscapine is an opium-derived kinder-gentler microtubule-modulating drug, currently in Phase I/II clinical trials for cancer chemotherapy. Here, we report the synthesis of four more potent di-substituted brominated derivatives of noscapine, 9-Br-7-OH-NOS (2), 9-Br-7-OCONHEt-NOS (3), 9-Br-7-OCONHBn-NOS (4), and 9-Br-7-OAc-NOS (5) and their chemotherapeutic efficacy on PC-3 and MDA-MB-231 cells. The four derivatives were observed to have higher tubulin binding activity than noscapine and significantly affect tubulin polymerization. The equilibrium dissociation constant (KD) for the interaction between tubulin and 2, 3, 4, 5 was found to be, 55±6µM, 44±6µM, 26±3µM, and 21±1µM respectively, which is comparable to parent analog. The effects of these di-substituted noscapine analogs on cell cycle parameters indicate that the cells enter a quiescent phase without undergoing further cell division. The varying biological activity of these analogs and bulk of substituent at position-7 of the benzofuranone ring system of the parent molecule was rationalized utilizing predictive in silico molecular modeling. Furthermore, the immunoblot analysis of protein lysates from cells treated with 4 and 5, revealed the induction of apoptosis and down-regulation of survivin levels. This result was further supported by the enhanced activity of caspase-3/7 enzymes in treated samples compared to the controls. Hence, these compounds showed a great potential for studying microtubule-mediated processes and as chemotherapeutic agents for the management of human cancers.

Abrogation of plasminogen activator inhibitor-1-vitronectin interaction ameliorates acute kidney injury in murine endotoxemia.

Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1-/-) or expressing a PAI-1-mutant (PAI-1R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1-/- and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1-/- mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.

Promoting microtubule assembly: A hypothesis for the functional significance of the +TIP network.

Regulation of microtubule (MT) dynamics is essential for many cellular processes, but the machinery that controls MT dynamics remains poorly understood. MT plus-end tracking proteins (+TIPs) are a set of MT-associated proteins that dynamically track growing MT ends and are uniquely positioned to govern MT dynamics. +TIPs associate with each other in a complex array of inter- and intra-molecular interactions known as the "+TIP network." Why do so many +TIPs bind to other +TIPs? Typical answers include the ideas that these interactions localize proteins where they are needed, deliver proteins to the cortex, and/or create regulatory pathways. We propose an additional and more mechanistic hypothesis: that +TIPs bind each other to create a superstructure that promotes MT assembly by constraining the structural fluctuations of the MT tip, thus acting as a polymerization chaperone.

Mechanism for the catastrophe-promoting activity of the microtubule destabilizer Op18/stathmin.

Regulation of microtubule dynamic instability is crucial for cellular processes, ranging from mitosis to membrane transport. Stathmin (also known as oncoprotein 18/Op18) is a prominent microtubule destabilizer that acts preferentially on microtubule minus ends. Stathmin has been studied intensively because of its association with multiple types of cancer, but its mechanism of action remains controversial. Two models have been proposed. One model is that stathmin promotes microtubule catastrophe indirectly, and does so by sequestering tubulin; the other holds that stathmin alters microtubule dynamics by directly destabilizing growing microtubules. Stathmin's sequestration activity is well established, but the mechanism of any direct action is mysterious because stathmin binds to microtubules very weakly. To address these issues, we have studied interactions between stathmin and varied tubulin polymers. We show that stathmin binds tightly to Dolastatin-10 tubulin rings, which mimic curved tubulin protofilaments, and that stathmin depolymerizes stabilized protofilament-rich polymers. These observations lead us to propose that stathmin promotes catastrophe by binding to and acting upon protofilaments exposed at the tips of growing microtubules. Moreover, we suggest that stathmin's minus-end preference results from interactions between stathmin's N terminus and the surface of α-tubulin that is exposed only at the minus end. Using computational modeling of microtubule dynamics, we show that these mechanisms could account for stathmin's observed activities in vitro, but that both the direct and sequestering activities are likely to be relevant in a cellular context. Taken together, our results suggest that stathmin can promote catastrophe by direct action on protofilament structure and interactions.

Performance evaluation of an algorithm for fast optimization of beam weights in anatomy-based intensity modulated radiotherapy.

This study aims to evaluate the performance of a new algorithm for optimization of beam weights in anatomy-based intensity modulated radiotherapy (IMRT). The algorithm uses a numerical technique called Gaussian-Elimination that derives the optimum beam weights in an exact or non-iterative way. The distinct feature of the algorithm is that it takes only fraction of a second to optimize the beam weights, irrespective of the complexity of the given case. The algorithm has been implemented using MATLAB with a Graphical User Interface (GUI) option for convenient specification of dose constraints and penalties to different structures. We have tested the numerical and clinical capabilities of the proposed algorithm in several patient cases in comparison with KonRad((R)) inverse planning system. The comparative analysis shows that the algorithm can generate anatomy-based IMRT plans with about 50% reduction in number of MUs and 60% reduction in number of apertures, while producing dose distribution comparable to that of beamlet-based IMRT plans. Hence, it is clearly evident from the study that the proposed algorithm can be effectively used for clinical applications.

Probing interactions between CLIP-170, EB1, and microtubules.

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other +TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of alpha-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both alpha-tubulin and beta-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both alpha-tubulin and beta-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the +TIP network.

Interactions between EB1 and microtubules: dramatic effect of affinity tags and evidence for cooperative behavior.

Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.