A comprehensive quality control and cost comparison study of branded and generic angiotensin receptor blockers.

This study was designed to assess both the quality and cost aspects of various branded and generic formulations of angiotensin receptor blockers, specifically Irbesartan, Losartan Potassium, Olmesartan Medoxomil, Telmisartan, and Valsartan. The collected samples underwent distinct quality evaluations using the methods outlined in different global Pharmacopoeias (British Pharmacopoeia/European Pharmacopoeia, Indian Pharmacopoeia and United States Pharmacopoeia). These drugs were characterized using Fourier-Transform Infrared Spectroscopy and Nuclear Magnetic Resonance techniques, while their quality and concentration were analysed using High Performance Liquid Chromatography. The release profile of the drugs was examined through dissolution testing. Additionally, a cost comparison analysis was carried out by determining the prevailing market prices of the drugs. The evaluated branded and generic angiotensin receptor blockers were found to meet the established standards for impurities, active drug content, and dissolution as set by these Pharmacopoeias, indicating their optimal quality. Notably, the generic drugs exhibited significantly lower costs compared to their branded counterparts. This study confirms that the quality of generic angiotensin receptor blockers is equivalent to that of their branded counterparts. Consequently, these findings support the practicality of utilizing generic drugs as a more economically sustainable and cost-effective approach to managing diseases, especially those of chronic nature.

Ensuring the quality of medicines in India: An update on the development, modernization, and harmonization of drug standards in the Indian Pharmacopoeia.

India has a sparkling pharmaceutical sector that holds a distinguished place by producing and supplying high-quality and affordable medicines across the globe. Ensuring the quality and safety of the marketed medicinal products is one of the most important components of the drug regulatory framework and assessment of the quality of medicines is usually achieved by referring to the public standards of the official Pharmacopoeia. In India, the Indian Pharmacopoeia (IP) is published at regular intervals to fulfill the requirements of the Drugs and Cosmetics Act, 1940 to ensure the quality of medicines being manufactured and/or marketed in India. The present article aims to provide an overview of the history of the IP, its standards-setting process, and the current status of monographs in the 9th edition of the IP 2022. Special focus is placed on the newly added and upgraded general chapters and monographs within the IP 2022. There are a total of 223 general chapters and 3152 drug monographs available under various categories in the IP 2022. This study also highlights a total of 92 new drug monograph additions and 412 monograph revisions in the IP 2022. It is anticipated that the standards laid down in the IP 2022 will play an imperative role in delivering quality medicines to patients within and outside India.

Anti-cancer activity of guggulsterone by modulating apoptotic markers: a systematic review and meta-analysis.

Background: Guggulsterone (pregna-4,17-diene-3,16-dione; C21H28O2) is an effective phytosterol isolated from the gum resin of the tree Commiphora wightii (Family Burseraceae) and is responsible for many of the properties of guggul. This plant is widely used as traditional medicine in Ayurveda and Unani system of medicine. It exhibits several pharmacological activities, such as anti-inflammatory, analgesic, antibacterial, anti-septic and anticancer. In this article, the activities of Guggulsterone against cancerous cells were determined and summarized. Methods: Using 7 databases (PubMed, PMC, Google Scholar, Science Direct, Scopus, Cochrane and Ctri.gov), the literature search was conducted since conception until June 2021. Extensive literature search yielded 55,280 studies from all the databases. A total of 40 articles were included in the systematic review and of them, 23 articles were included in the meta-analysis.The cancerous cell lines used in the studies were for pancreatic cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, cholangiocarcinoma, oesophageal adenocarcinoma, prostrate cancer, colon cancer, breast cancer, gut derived adenocarcinoma, gastric cancer, colorectal cancer, bladder cancer, glioblastoma, histiocytic leukemia, acute myeloid leukemia and non-small cell lung cancer. The reliability of the selected studies was assessed using ToxRTool. Results: Based on this review, guggulsterone significantly affected pancreatic cancer (MiaPaCa-2, Panc-1, PC-Sw, CD18/HPAF, Capan1, PC-3), hepatocellular carcinoma (Hep3B, HepG2, PLC/PRF/5R), head and neck squamous cell carcinoma (SCC4, UM-22b, 1483), cholangiocarcinoma (HuCC-T1, RBE, Sk-ChA-1, Mz-ChA-1) and oesophageal adenocarcinoma (CP-18821, OE19), prostrate cancer (PC-3), colon cancer (HT-29), breast cancer (MCF7/DOX), gut derived adenocarcinoma (Bic-1), gastric cancer (SGC-7901), colorectal cancer (HCT116), bladder cancer (T24, TSGH8301), glioblastoma (A172, U87MG, T98G), histiocytic leukemia (U937), acute myeloid leukemia (HL60, U937) and non-small cell lung cancer (A549, H1975) by inducing apoptotic pathways, inhibiting cell proliferation, and regulating the expression of genes involved in apoptosis. Guggulsterone is known to have therapeutic and preventive effects on various categories of cancers. It can inhibit the progression of tumors and can even reduce their size by inducing apoptosis, exerting anti-angiogenic effects, and modulating various signaling cascades. In vitro studies reveal that Guggulsterone inhibits and suppresses the proliferation of an extensive range of cancer cells by decreasing intrinsic mitochondrial apoptosis, regulating NF-kB/STAT3/ß-Catenin/PI3K/Akt/CHOP pathway, modulating the expression of associated genes/proteins, and inhibiting angiogenesis. Furthermore, Guggulsterone reduces the production of inflammatory markers, such as CDX2 and COX-2. The other mechanism of the Guggulsterone activity is the reversal of P-glycoprotein-mediated multidrug resistance. Twenty three studies were selected for meta-analysis following the PRISMA statements. Fixed effect model was used for reporting the odds ratio. The primary endpoint was percentage apoptosis. 11 of 23 studies reported the apoptotic effect at t = 24 h and pooled odds ratio was 3.984 (CI 3.263 to 4.865, p < 0.001). 12 studies used Guggulsterone for t > 24 h and the odds ratio was 11.171 (CI 9.148 to 13.643, 95% CI, p < 0.001). The sub-group analysis based on cancer type, Guggulsterone dose, and treatment effects. Significant alterations in the level of apoptotic markers were reported by Guggulsterone treatment. Conclusion: This study suggested that Guggulsterone has apoptotic effects against various cancer types. Further investigation of its pharmacological activity and mechanism of action should be conducted. In vivo experiments and clinical trials are required to confirm the anticancer activity.

Pharmacopoeia roles and responses: A systemic resilience approach to COVID-19 pandemic.

The Coronavirus Disease (COVID-19) is sweeping around the world at a rapid pace resulting in severe health crises across the globe. The pandemic condition has forced the government, regulatory authorities, bio/pharmaceutical industry, and healthcare system to take novel measures to address the crisis. The race for development of medicines and vaccines for treatment of COVID-19 is well under way and regulatory authorities are making efforts to safely deliver it into hands of public. As ever, pharmacopoeias played an active role in providing a framework of standards for the development, manufacturing, and quality of life-saving COVID-19 related medicines. The COVID-19 crisis has compelled the pharmacopoeias to redefine their role and show unprecedented levels of flexibility in extending their services to the stakeholders, developing new drug standards, and simultaneously ensuring the safety of their staff. During this pandemic, pharmacopoeias operated in a triangular chain system with regulators and pharmaceutical manufacturers to evaluate potential products for treatment of COVID-19. The present article provides an insight on the roles, challenges, and responses of the pharmacopoeias to deal with the current situation due to COVID-19 and emphasizes on new opportunities for collaborations to set standards for COVID-19 related drugs.

Comparative Study of Adverse Drug Reactions Associated with Filgrastim and Pegfilgrastim Using the EudraVigilance Database.

The primary prophylaxis with filgrastim (FIL) and pegfilgrastim (PEG-F) is recommended to decrease the severity of chemotherapy-induced neutropenia (CIN). The commonly reported adverse drug reactions (ADRs) with FIL and PEG-F is bone pain. ADRs pertaining to FIL and PEG-F were extracted from the European EudraVigilance (EV) database. The Individual Case Safety Reports (ICSRs) obtained from EV database that reported FIL and PEG-F as the suspected drug were analyzed. Registered ADRs (from the groups "General disorders and administration site conditions", "Blood and lymphatic system disorders", "Musculoskeletal and connective tissue disorders" and "Investigations") for FIL and PEG-F were collected from EV database from 2007 to 5 June 2021. The reporting odds ratio (ROR) was used to calculate ICSRs with most common ADRs related to FIL and PEG-F. A total of 17,403 ICSRs described the incidence of most common ADRs of FIL and PEG-F. The commonly reported ADRs for both drugs were pyrexia, bone pain, back pain, neutropenia and febrile neutropenia. The odds ratio of ICSRs belonging to the System Organ Class (SOC) "Investigations" (ROR 1.01 (CI 0.93-1.10)) revealed no significant difference in FIL and PEG-F. However, for the SOCs (General disorders and administration site conditions" and "Musculoskeletal and connective tissue disorders" ((ROR 1.14 (CI 1.06-1.21); ROR 1.21 (CI 1.18-1.32), respectively), an increased reporting probability with PEG-F was found. The authors reported a lower reporting probability for the SOC "Blood and lymphatic system disorders" for FIL versus PEG-F (ROR 0.75 (CI 0.70-0.80)). Our results have demonstrated that the occurrence of bone pain was similar with FIL and PEG-F. For the incidence of pyrexia and back pain, PEG-F was associated with a higher reporting probability as compared to FIL. However, the incidence of neutropenia and febrile neutropenia was higher in FIL compared to PEG-F. Further evaluation of data from real life is needed.

Efficacy and Safety of Filgrastim and Its Biosimilars to Prevent Febrile Neutropenia in Cancer Patients: A Prospective Study and Meta-Analysis.

Background: The aim of this review and meta-analysis was to identify, assess, meta-analyze and summarize the comparative effectiveness and safety of filgrastim in head-to-head trials with placebo/no treatment, pegfilgrastim (and biosimilar filgrastim to update advances in the field. Methods: The preferred reporting items for systematic reviews and meta-analyses PRISMA statement were applied, and a random-effect model was used. Primary endpoints were the rate and duration of grade 3 or 4 neutropenia, and an incidence rate of febrile neutropenia. Secondary endpoints were time to absolute neutrophil count ANC recovery, depth of ANC nadir (lowest ANC), neutropenia-related hospitalization and other neutropenia-related complications. For filgrastim versus biosimilar filgrastim comparison, the primary efficacy endpoint was the mean difference in duration of severe neutropenia DSN. Results: A total of 56 studies were considered that included data from 13,058 cancer patients. The risk of febrile neutropenia in filgrastim versus placebo/no treatment was not statistically different. The risk ratio for febrile neutropenia was 0.58, a 42% reduction in favor of filgrastim. The most reported adverse event with FIL was bone pain. For pegfilgrastim versus filgrastim, no statistically significant difference was noted. The risk ratio was 0.90 (95% CI 0.67 to 1.12). The overall difference in duration of severe neutropenia between filgrastim and biosimilar filgrastim was not statistically significant. The risk ratio was 1.03 (95% CI 0.93 to 1.13). Conclusions: Filgrastim was effective and safe in reducing febrile neutropenia and related complications, compared to placebo/no treatment. No notable differences were found between pegfilgrastim and filgrastim in terms of efficacy and safety. However, a similar efficacy profile was observed with FIL and its biosimilars.

Severe cutaneous adverse reactions in Asians: Trends observed in culprit anti-seizure medicines using VigiBase®.

PURPOSE: Diverse ethnic genetic populations display variability in the risk regarding anti-seizure medicine (ASM)-induced severe cutaneous adverse reactions (SCARs). However, clinical and epidemiological data on ASM-induced SCARs in Asians is limited. METHODS: We conducted a retrospective, post-market study until April 30, 2020 using VigiBase® for demographic characteristics, causative ASMs, complications and mortality. The study included adverse events as classified by Standardized Medical Dictionary for Regulatory Activities (MedDRA) queries of SCARs, mainly Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), drug rash with eosinophilia and systemic symptoms (DRESS), and SJS/TEN overlap reported for ASMs. RESULTS: A total of 694,811 adverse events were reported across the world while using ASMs. Of this, skin and subcutaneous tissue adverse events were 122,885 (17.6%). Among ASM-induced skin and subcutaneous tissue adverse events, SJS, TEN, DRESS and SJS/TEN overlap represented 11,181 (9.1%), 3,645 (3.0%), 5,106 (4.1%) and 6 (0.004%) cases, respectively. Female SJS/TEN/DRESS patients were 54.1%, and 75% of them were adults (>18Y). Nearly 64% of the ASM-induced SCARs were serious and culminated in death (3.5%), life-threatening conditions (11.5%), and hospitalization/prolonged hospitalization (43.5%) of patients on ASM therapy. Carbamazepine (31.6%), phenytoin (29.6%), lamotrigine (24.3%), valproic acid (6.4%) and phenobarbital (5.7%) are the most commonly used ASMs linked with SCARs. ASMs associated with significantly higher risk of SCARs in Asians were carbamazepine [n = 3265, ROR 3.55 (95% CI 3.38-3.72, P < 0.0001)], lamotrigine [n = 1253, ROR 3.90 (95% CI 3.63-4.18, P < 0.0001)], gabapentin [n = 85, ROR 3.58 (95% CI 2.79-4.60, P < 0.0001)], pregabalin [n = 68, ROR 3.16 (95% CI 2.40-4.16, P < 0.0001)], clonazepam [n = 53, ROR 3.19 (95% CI 2.31-4.41, P < 0.0001)], lorazepam [n = 31, ROR 3.07 (95% CI 2.06-4.59, P < 0.0001)] and acetazolamide [n = 28, ROR 3.90 (95% CI 2.45-6.21, P < 0.0001)]. CONCLUSION: Based on our study, carbamazepine, lamotrigine, gabapentin, pregabalin, clonazepam, lorazepam, and acetazolamide are the most common causative ASMs for SCARs in the Asian population.

Drug delivery device for the inner ear: ultra-sharp fully metallic microneedles.

Drug delivery into the inner ear is a significant challenge due to its inaccessibility as a fluid-filled cavity within the temporal bone of the skull. The round window membrane (RWM) is the only delivery portal from the middle ear to the inner ear that does not require perforation of bone. Recent advances in microneedle fabrication enable the RWM to be perforated safely with polymeric microneedles as a means to enhance the rate of drug delivery from the middle ear to the inner ear. However, the polymeric material is not biocompatible and also lacks the strength of other materials. Herein we describe the design and development of gold-coated metallic microneedles suitable for RWM perforation. When developing microneedle technology for drug delivery, we considered three important general attributes: (1) high strength and ductility material, (2) high accuracy and precision of fabrication, and (3) broad design freedom. We developed a hybrid additive manufacturing method using two-photon lithography and electrochemical deposition to fabricate ultra-sharp gold-coated copper microneedles with these attributes. We refer to the microneedle fabrication methodology as two-photon templated electrodeposition (2PTE). We demonstrate the use of these microneedles by inducing a perforation with a minimal degree of trauma in a guinea pig RWM while the microneedle itself remains undamaged. Thus, this microneedle has the potential literally of opening the RWM for enhanced drug delivery into the inner ear. Finally, the 2PTE methodology can be applied to many different classes of microneedles for other drug delivery purposes as well the fabrication of small scale structures and devices for non-medical applications. Graphical Abstract Fully metallic ultra-sharp microneedle mounted at end of a 24-gauge stainless steel blunt syringe needle tip: (left) Size of microneedle shown relative to date stamp on U.S. one-cent coin; (right) Perforation through guinea pig round window membrane introduced with microneedle.

PGE2 deficiency predisposes to anaphylaxis by causing mast cell hyperresponsiveness.

BACKGROUND: Reduced levels of prostaglandin E2 (PGE2) contribute to aspirin-induced hypersensitivity. COX inhibitors are also frequent cofactors in anaphylaxis. Whether alterations in the PGE2 system contribute to anaphylaxis independently of COX inhibitor intake is unclear. OBJECTIVE: Our aim was to test the hypothesis that relative PGE2 deficiency predisposes to anaphylaxis. METHODS: Sera from 48 patients with anaphylaxis and 27 healthy subjects were analyzed for PGE2 levels and correlated against severity; 9α,11ß-PGF2 and PGI2 metabolites were measured for control purposes. PGE2 stabilization by 15-hydroxyprostaglandin dehydrogenase inhibitor or EP2 or EP4 receptor agonists were used in a murine model of passive systemic anaphylaxis. FcεRI-triggered mediator release was determined in bone marrow-derived cultured mast cells (MCs) and human skin-derived MCs. Signaling was studied by Western blot analysis. RESULTS: Patients with anaphylaxis were characterized by markedly reduced PGE2 levels vis-à-vis healthy subjects, whereas prostacyclin metabolite levels were diminished only weakly, and 9α,11ß-PGF2 levels conversely increased. PGE2 was negatively correlated with severity. Lower PGE2 levels and higher susceptibility to anaphylaxis were also found in C57BL/6 mice vis-à-vis in Balb/c mice. Stabilization of PGE2 level by 15-hydroxyprostaglandin dehydrogenase inhibitor protected mice against anaphylaxis. Exogenous PGE2 attenuated bone marrow-derived cultured MC activation through EP2 and EP4 receptors. EP2 and EP4 agonism also curbed FcεRI-mediated degranulation of human MCs. Mechanistically, PGE2 interfered with the phosphorylation of phospholipase C gamma-1 and extracellular signal-regulated kinase. CONCLUSIONS: Homeostatic levels of PGE2 attenuate MC activation via EP2/EP4 and protect against anaphylaxis. Relative deficiency of PGE2 predisposes to anaphylaxis in humans and mice, whereas PGE2 stabilization protects against anaphylactic reactions.

Towards a comprehensive safety understanding of granulocyte-colony stimulating factor biosimilars in treating chemotherapy associated febrile neutropenia: Trends from decades of data.

Filgrastim, a biopharmaceutical listed on WHO model list of essential medicines, was approved in USA in 1991 for patients with non-myeloid malignancies associated with severe neutropenia and fever. Several filgrastim biosimilars have now been approved in USA, Europe and elsewhere since 2008, based on the reference product which has lost patent exclusivity; however their immunogenicity and safety is controversial. We conducted a retrospective, post market study between 1991 and May 2018 using VigiBase®. The study included all adverse events with case reports ≥150. Overall, 11,183 adverse drugs reaction reports were identified during observation period; of which 5764; 51.5% reports concerned to Neupogen®, the originator, and rest consists of Leucostim® (N = 680), Zarzio® (N = 622), Grasin® (N = 545), Nivestim® (N = 359) and Tevagrastim® (N = 152) biosimilars. When compared with the originator, Grasin® was associated with higher reporting of pyrexia (11.5% vs 7.9%, ROR 1.52, IC025 1.12), myalgia (37% vs 2.2%, ROR 25.94, IC025 2.11) and back pain (11.3% vs 4%, ROR 3.09, IC025 2.32). Zarzio® was associated with increased reporting of arthralgia (4.5% vs 2.9%, ROR 1.59, IC025 1.25) and neutropenia (11.4% vs 4%, ROR 2.59, IC025 3.07). Bone pain was reported more often with Nivestim® (14.4% vs 8.3%, ROR 1.87, IC025 5.30). Drug ineffectiveness was reported in cases with Zarzio® (35.9%), Nivestim® (19.4%) and Tevagrastim® (42.2%). Authors observed significant differences among originator and biosimilars in particular to efficacy, adverse events reported and time to onset of occurrences. Large epidemiologic studies are needed to further confirm these finding and provide additional insights.

Peptide-based therapeutics: quality specifications, regulatory considerations, and prospects.

Exquisite selectivity, remarkable efficacy, and minimal toxicity are key attributes inherently assigned to peptides, resulting in increased research interest from the pharmaceutical industry in peptide-based therapeutics (PbTs). Pharmacopoeias develop authoritative standards for PbT by providing standard specifications and test methods. Nevertheless, a lack of harmonization in test procedures adopted for PbT in the latest editions of Pharmacopoeias has been observed. Adoption of a harmonized monograph could increase further the interest of the global pharmaceutical industry in PbTs. Here, we provide an overview of pharmacopoeial methodologies and specifications commonly observed in PbT monographs and highlight the main differences among the pharmacopoeias in terms of the active pharmaceutical ingredients that they focus on. We also address the prospects for PbTs to mature as a new therapeutic niche.

Veterinary herbal medicines in India.

India has a rich and diversified flora. It is seen that synthetic drugs could pose serious problems, are toxic and costly. In contrast to this, herbal medicines are relatively nontoxic, cheaper and are eco-friendly. Moreover, the people have used them for generations. They have also been used in day-to-day problems of healthcare in animals. 25% of the drugs prescribed worldwide come from plants. Almost 75% of the medicinal plants grow naturally in different states of India. These plants are known to cure many ailments in animals like poisoning, cough, constipation, foot and mouth disease, dermatitis, cataract, burning, pneumonia, bone fractures, snake bites, abdominal pains, skin diseases etc. There is scarce review of such information (veterinary herbals) in the literature. The electronic and manual search was made using various key words such as veterinary herbal, ethno-veterinary medicines etc. and the content systematically arranged. This article deals with the comprehensive review of 45 medicinal plant species that are official in Indian Pharmacopoeia (IP) 2014. The botanical names, family, habitat, plant part used and pharmacological actions, status in British Pharmacopoeia 2014, USP 36 are mentioned. Also, a relationship between animal and human dose, standardization and regulatory aspects of these selected veterinary herbals are provided.

Implementing the Principle of the 3 Rs Through the Indian Pharmacopoeia.

Quality and safety tests are required for regulatory approval of drugs and pharmaceuticals in the country to guarantee minimum safety standards, and most of these tests include animal usage. In the case of biological medicines, these safety and quality tests have to be performed on a batch-to-batch basis and require a large number of animals. Russell and Burch's 1959 principle of the 3 Rs- replacement, reduction, and refinement-is now being increasingly adopted worldwide, and various national and international pharmacopoeias have taken initiatives to safeguard animals. This article details the Indian Pharmacopoeia Commission's initiative to implement the 3 Rs through the Indian Pharmacopoeia. Explored are the deletion of animal tests, such as the abnormal toxicity test at final lot for biologicals; the replacement of in vivo methods by in vitro methods; the reduction in the number of animals used where deletion of the animal test is not possible; and the refinement of tests to cause minimal suffering to the animals. In Indian Pharmacopoeia 2014, pyrogen testing using rabbits has been replaced by the bacterial endotoxin test in the majority of biological monographs-keeping in view international trends and, especially for vaccine monographs, validated in vitro tests such as the bacterial endotoxin test as an alternative to the pyrogen test where justified and authorized. Steps are taken for introducing a single-dilution assay for the potency testing of diphtheria and tetanus vaccine (adsorbed) with the aim of minimizing number of animals used. The justified and authorized use of animals in drug manufacturing, analytic laboratories, and research will not only help in the expedited development/production of drugs but also be useful in protecting and promoting animal health.

LocDB: experimental annotations of localization for Homo sapiens and Arabidopsis thaliana.

LocDB is a manually curated database with experimental annotations for the subcellular localizations of proteins in Homo sapiens (HS, human) and Arabidopsis thaliana (AT, thale cress). Currently, it contains entries for 19,604 UniProt proteins (HS: 13,342; AT: 6262). Each database entry contains the experimentally derived localization in Gene Ontology (GO) terminology, the experimental annotation of localization, localization predictions by state-of-the-art methods and, where available, the type of experimental information. LocDB is searchable by keyword, protein name and subcellular compartment, as well as by identifiers from UniProt, Ensembl and TAIR resources. In comparison to other public databases, LocDB as a resource adds about 10,000 experimental localization annotations for HS proteins and ∼900 for AS proteins. Over 40% of the proteins in LocDB have multiple localization annotations providing a better platform for development of new multiple localization prediction methods with higher coverage and accuracy. Links to all referenced databases are provided. LocDB will be updated regularly by our group (available at: http://www.rostlab.org/services/locDB).

Bioinformatics predictions of localization and targeting.

One of the major challenges in the post-genomic era with hundreds of genomes sequenced is the annotation of protein structure and function. Computational predictions of subcellular localization are an important step toward this end. The development of computational tools that predict targeting and localization has, therefore, been a very active area of research, in particular since the first release of the groundbreaking program PSORT in 1991. The most reliable means of annotating protein structure and function remains homology-based inference, i.e. the transfer of experimental annotations from one protein to its homologs. However, annotations about localization demonstrate how much can be gained from advanced machine learning: more proteins can be annotated more reliably. Contemporary computational tools for the annotation of protein targeting include automatic methods that mine the textual information from the biological literature and molecular biology databases. Some machine learning-based methods that accurately predict features of sorting signals and that use sequence-derived features to predict localization have reached remarkable levels of performance. Sustained prediction accuracy has increased by more than 30 percentage points over the last decade. Here, we review some of the most recent methods for the prediction of subcellular localization and protein targeting that contributed toward this breakthrough.

Characterizing positive and negative selection and their phylogenetic effects.

Total evidence and the use of large datasets to overcome uncertainty are the state of the art in systematic analysis. This assumes that the only true phylogenetic signal is ancestry and that functional, structural, and other factors will not add an alternative signal. Using gene families, where individual codon positions were sorted into bins based upon average-pairwise dN/dS ratio, we show that standard, common phylogenetic methods that were designed for stochastic, neutral, site-independent processes, generate less robust phylogenetic signal for bins with strong negative or positive selection. This was true for phylogenetic reconstruction with parsimony, distance, and likelihood methods. Further, we present a case for the potential existence of systematic functional or structural signal that competes with ancestral signal. For the example of positive selection, we simulate the evolution of sequences through three dimensional lattice constructs with folding constraint and changing binding functionality and show that total evidence for these lattice genes presents trees with functional signal, but that the neutral synonymous sites in these genes show the true ancestral signal. In this case, sequence convergence is promoted by functional convergence.

Evolution after gene duplication: models, mechanisms, sequences, systems, and organisms.

Gene duplication is postulated to have played a major role in the evolution of biological novelty. Here, gene duplication is examined across levels of biological organization in an attempt to create a unified picture of the mechanistic process by which gene duplication can have played a role in generating biodiversity. Neofunctionalization and subfunctionalization have been proposed as important processes driving the retention of duplicate genes. These models have foundations in population genetic theory, which is now being refined by explicit consideration of the structural constraints placed upon genes encoding proteins through physical chemistry. Further, such models can be examined in the context of comparative genomics, where an integration of gene-level evolution and species-level evolution allows an assessment of the frequency of duplication and the fate of duplicate genes. This process, of course, is dependent upon the biochemical role that duplicated genes play in biological systems, which is in turn dependent upon the mechanism of duplication: whole genome duplication involving a co-duplication of interacting partners vs. single gene duplication. Lastly, the role that these processes may have played in driving speciation is examined.

Evaluation of models for the evolution of protein sequences and functions under structural constraint.

In the field of evolutionary structural genomics, methods are needed to evaluate why genomes evolved to contain the fold distributions that are observed. In order to study the effects of population dynamics in the evolved genomes we need fast and accurate evolutionary models which can analyze the effects of selection, drift and fixation of a protein sequence in a population that are grounded by physical parameters governing the folding and binding properties of the sequence. In this study, various knowledge-based, force field, and statistical methods for protein folding have been evaluated with four different folds: SH2 domains, SH3 domains, Globin-like, and Flavodoxin-like, to evaluate the speed and accuracy of the energy functions. Similarly, knowledge-based and force field methods have been used to predict ligand binding specificity in SH2 domain. To demonstrate the applicability of these methods, the dynamics of evolution of new binding capabilities by an SH2 domain is demonstrated.

Subfunctionalization of duplicated genes as a transition state to neofunctionalization.

BACKGROUND: Gene duplication has been suggested to be an important process in the generation of evolutionary novelty. Neofunctionalization, as an adaptive process where one copy mutates into a function that was not present in the pre-duplication gene, is one mechanism that can lead to the retention of both copies. More recently, subfunctionalization, as a neutral process where the two copies partition the ancestral function, has been proposed as an alternative mechanism driving duplicate gene retention in organisms with small effective population sizes. The relative importance of these two processes is unclear. RESULTS: A set of lattice model genes that fold and bind to two peptide ligands with overlapping binding pockets, but not a third ligand present in the cell was designed. Each gene was duplicated in a model haploid species with a small constant population size and no recombination. One set of models allowed subfunctionalization of binding events following duplication, while another set did not allow subfunctionalization. Modeling under such conditions suggests that subfunctionalization plays an important role, but as a transition state to neofunctionalization rather than as a terminal fate of duplicated genes. There is no apparent selective pressure to maintain redundancy. CONCLUSION: Subfunctionalization results in an increase in the preservation of duplicated gene copies, including those that are neofunctionalized, but never represents a substantial fraction of duplicate gene copies at any evolutionary time point and ultimately leads to neofunctionalization of those preserved copies. This conclusion also may reflect changes in gene function after duplication with time in real genomes.