Long-Term Evaluation of Retinal Morphology and Function in Rosa26-Cas9 Knock-In Mice.

The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.

Membrane skeletal association and post-translational allosteric regulation of Toxoplasma gondii GAPDH1.

When Toxoplasma gondii egresses from the host cell, glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1), which is primary a glycolysis enzyme but actually a quintessential multifunctional protein, translocates to the unique cortical membrane skeleton. Here, we report the 2.25 Å resolution crystal structure of the GAPDH1 holoenzyme in a quaternary complex providing the basis for the molecular dissection of GAPDH1 structure-function relationships Knockdown of GAPDH1 expression and catalytic site disruption validate the essentiality of GAPDH1 in intracellular replication but we confirmed that glycolysis is not strictly essential. We identify, for the first time, S-loop phosphorylation as a novel, critical regulator of enzymatic activity that is consistent with the notion that the S-loop is critical for cofactor binding, allosteric activation and oligomerization. We show that neither enzymatic activity nor phosphorylation state correlate with the ability to translocate to the cortex. However, we demonstrate that association of GAPDH1 with the cortex is mediated by the N-terminus, likely palmitoylation. Overall, glycolysis and cortical translocation are functionally decoupled by post-translational modifications.

Analysis of sampling techniques for imbalanced data: An n = 648 ADNI study.

Many neuroimaging applications deal with imbalanced imaging data. For example, in Alzheimer's Disease Neuroimaging Initiative (ADNI) dataset, the mild cognitive impairment (MCI) cases eligible for the study are nearly two times the Alzheimer's disease (AD) patients for structural magnetic resonance imaging (MRI) modality and six times the control cases for proteomics modality. Constructing an accurate classifier from imbalanced data is a challenging task. Traditional classifiers that aim to maximize the overall prediction accuracy tend to classify all data into the majority class. In this paper, we study an ensemble system of feature selection and data sampling for the class imbalance problem. We systematically analyze various sampling techniques by examining the efficacy of different rates and types of undersampling, oversampling, and a combination of over and undersampling approaches. We thoroughly examine six widely used feature selection algorithms to identify significant biomarkers and thereby reduce the complexity of the data. The efficacy of the ensemble techniques is evaluated using two different classifiers including Random Forest and Support Vector Machines based on classification accuracy, area under the receiver operating characteristic curve (AUC), sensitivity, and specificity measures. Our extensive experimental results show that for various problem settings in ADNI, (1) a balanced training set obtained with K-Medoids technique based undersampling gives the best overall performance among different data sampling techniques and no sampling approach; and (2) sparse logistic regression with stability selection achieves competitive performance among various feature selection algorithms. Comprehensive experiments with various settings show that our proposed ensemble model of multiple undersampled datasets yields stable and promising results.

Daunorubicin forms a specific complex with a secreted serine protease of Streptomyces peucetius.

Daunorubicin forms specific complex with an extracellular protease in the Streptomyces peucetius culture. The drug-protein complex co-migrates in non-denaturing PAGE as a red band. De novo peptide sequencing by nano-LC-ESI-MS/MS and MASCOT analysis identified the daunorubicin binding protein as serine protease precursor. The same protease precursor was purified sans the daunorubicin, from the mutant named ΔDPSAmut, which is deficient in daunorubicin production. Daunorubicin was added to ΔDPSAmut culture and the protease readily formed the daunorubicin-protease complex. Ability of serine protease precursor to form a selective complex with daunorubicin was confirmed by this study. Selective binding of protease to daunorubicin was seen as self-resistance determinant for the organism to survive toxic levels of the drug outside the cell. Daunorubicin-protease complex placed on S. peucetius lawn did not produce clearing zone around it, whereas daunorubicin purified from the complex did produce the clearing zone. Thereby it is concluded that the protease sequesters daunorubicin to prevent its entry into cells. Sequestration of daunorubicin by extracellular protease helps the organism to maintain a steady state sub-inhibitory level of drug around the cells. A new self-resistance determinant is reported here.

Unraveling Histone Loss in Aging and Senescence.

As the global population experiences a notable surge in aging demographics, the need to understand the intricate molecular pathways exacerbated by age-related stresses, including epigenetic dysregulation, becomes a priority. Epigenetic mechanisms play a critical role in driving age-related diseases through altered gene expression, genomic instability, and irregular chromatin remodeling. In this review, we focus on histones, a central component of the epigenome, and consolidate the key findings of histone loss and genome-wide redistribution as fundamental processes contributing to aging and senescence. The review provides insights into novel histone expression profiles, nucleosome occupancy, disruptions in higher-order chromatin architecture, and the emergence of noncanonical histone variants in the aging cellular landscape. Furthermore, we explore the current state of our understanding of the molecular mechanisms of histone deficiency in aging cells. Specific emphasis is placed on highlighting histone degradation pathways in the cell and studies that have explored potential strategies to mitigate histone loss or restore histone levels in aging cells. Finally, in addressing future perspectives, the insights gained from this review hold profound implications for advancing strategies that actively intervene in modulating histone expression profiles in the context of cellular aging and identifying potential therapeutic targets for alleviating a multitude of age-related diseases.

Comparison of Analgesic Efficacy of Ultrasound Guided PEC II Block Using Dexamethasone as an Adjuvant to Ropivacaine Versus Plain Ropivacaine in Patients Undergoing Modified Radical Mastectomy: A Double-Blind, Randomized Controlled Trial.

INTRODUCTION:  Pain after breast cancer surgery is one of the main reasons for postoperative morbidity and pulmonary complications leading to increased hospital stay. Therefore, effective management of postoperative pain becomes necessary to alleviate patients' suffering and allow early mobilization and hospital discharge. Traditionally, opioids have been used to manage perioperative pain but they are associated with side effects. So, an opioid-sparing multimodal analgesia approach is used nowadays. Ultrasound-guided pectoral type-II (PEC II) block is increasingly being used to address acute postoperative pain after breast cancer surgery. However, to date, not many studies have been done regarding prolonging the duration of analgesia of PEC II blocks for postoperative pain relief in patients undergoing modified radical mastectomy (MRM). So, we undertook this study to compare the analgesic efficacy of PEC II block using dexamethasone as an adjuvant to ropivacaine versus plain ropivacaine in patients undergoing MRM. METHODS:  After obtaining approval from the institute ethics committee and written informed consent from the patients, this prospective, double-blind, parallel group, randomized controlled study was carried out at All India Institute of Medical Sciences (AIIMS) Raipur, from March 2021 to March 2022. Sixty-four female patients, aged 18 years and above, belonging to the American Society of Anesthesiologists, physical status I, II, and III, undergoing unilateral, elective MRM under general anesthesia, were randomly allocated to two groups A and B, with 32 patients in each to receive 30 mL of 0.25% ropivacaine plus 2 mL (8 mg) of dexamethasone and 30 mL of 0.25% ropivacaine plus 2 mL of normal saline, respectively. The primary outcome measure was total opioid consumption till 12 hours postoperatively. Secondary outcome measures were the difference in pain scores based on the numeric rating scale till 12 hours postoperatively, post-operative sedation scores, the incidence of postoperative nausea vomiting (PONV), and other adverse events (if any). RESULTS: The mean (SD) of morphine (mg) consumed intraoperatively was 5.50 (1.05) and 5.95 (0.86) with P = 0.033 and that consumed postoperatively was 1.00 (0.00) and 1.69 (0.93) with P <0.001 in group A and B respectively, with morphine consumption being higher in the group. The difference in the NRS score for pain at rest was statistically significant at 2 h (P=0.030), 4 h (P=0.004), 6 h (P=0.002), and, 12 h (P=0.039) time points with the score being higher in group B. The groups were comparable in terms of postoperative sedation score (P > 0.05) and incidence of postoperative nausea and vomiting. None of the patients in group A and 6.2% of the patients in group B had nausea (P = 0.492). None of the patients in either of the groups had vomiting. No other complication occurred during the entire study in either of the groups. CONCLUSION: In comparison to plain ropivacaine, the addition of dexamethasone as an adjuvant to ropivacaine for PEC II block in patients undergoing MRM significantly reduced perioperative opioid consumption and postoperative NRS scores. No significant change was noted in terms of postoperative sedation score, incidence of PONV, and other side effects between the groups. Therefore, we conclude that the analgesic efficacy of US-guided PEC II block using dexamethasone, as an adjuvant to ropivacaine is superior to that of plain ropivacaine in patients undergoing MRM.

Histone deficiency and hypoacetylation in the aging retinal pigment epithelium.

Histones serve as a major carrier of epigenetic information in the form of post-translational modifications which are vital for controlling gene expression, maintaining cell identity, and ensuring proper cellular function. Loss of histones in the aging genome can drastically impact the epigenetic landscape of the cell leading to altered chromatin structure and changes in gene expression profiles. In this study, we investigated the impact of age-related changes on histone levels and histone acetylation in the retinal pigment epithelium (RPE) and retina of mice. We observed a global reduction of histones H1, H2A, H2B, H3, and H4 in aged RPE/choroid but not in the neural retina. Transcriptomic analyses revealed significant downregulation of histones in aged RPE/choroid including crucial elements of the histone locus body (HLB) complex involved in histone pre-mRNA processing. Knockdown of HINFP, a key HLB component, in human RPE cells induced histone loss, senescence, and the upregulation of senescence-associated secretory phenotype (SASP) markers. Replicative senescence and chronological aging in human RPE cells similarly resulted in progressive histone loss and acquisition of the SASP. Immunostaining of human retina sections revealed histone loss in RPE with age. Acetyl-histone profiling in aged mouse RPE/choroid revealed a specific molecular signature with loss of global acetyl-histone levels, including H3K14ac, H3K56ac, and H4K16ac marks. These findings strongly demonstrate histone loss as a unique feature of RPE aging and provide critical insights into the potential mechanisms linking histone dynamics, cellular senescence, and aging.

Molecular docking study of conformational polymorph: building block of crystal chemistry.

Two conformational polymorphs of novel 2-[2-(3-cyano-4,6-dimethyl-2-oxo-2H-pyridin-1-yl)-ethoxy]-4,6-dimethyl nicotinonitrile have been developed. The crystal structure of both polymorphs (1a and 1b) seems to be stabilized by weak interactions. A difference was observed in the packing of both polymorphs. Polymorph 1b has a better binding affinity with the cyclooxygenase (COX-2) receptor than the standard (Nimesulide).

Environmental Factors Affecting Monoterpene Emissions from Terrestrial Vegetation.

Monoterpenes are volatile organic compounds that play important roles in atmospheric chemistry, plant physiology, communication, and defense. This review compiles the monoterpene emission flux data reported for different regions and plant species and highlights the role of abiotic environmental factors in controlling the emissions of biogenic monoterpenes and their emission fluxes for terrestrial plant species (including seasonal variations). Previous studies have demonstrated the role and importance of ambient air temperature and light in controlling monoterpene emissions, likely contributing to higher monoterpene emissions during the summer season in temperate regions. In addition to light and temperature dependence, other important environmental variables such as carbon dioxide (CO2), ozone (O3), soil moisture, and nutrient availability are also known to influence monoterpene emissions rates, but the information available is still limited. Throughout the paper, we identify knowledge gaps and provide recommendations for future studies.

A Rare Case of Giant Occipital Encephalocele With Thoracic Myelomeningocele: An Anesthetic Conundrum.

Encephalocele and myelomeningocele are congenital defects in the cranium and spine with herniation of contents into an extracranial and extraspinal sac, respectively. The occurrence of encephalocele and myelomeningocele in the same patient has rarely been described in the literature. The anesthetic management of such cases is associated with multiple challenges, which include difficulty in securing the airway, prone positioning, blood loss, electrolyte imbalance, hypothermia, cardiorespiratory disturbances, and perioperative care. The main aims are, to prevent hemodynamic fluctuations and excessive pressure on the sac to avoid premature rupture and manage a possible difficult airway due to the head and neck mass. We report such a rare case to highlight and share our experiences faced during perioperative management of a giant vascular occipital encephalocele with impending rupture and thoracic myelomeningocele requiring surgical excision and repair. Previous similar case reports were also reviewed, and potential perioperative complications were discussed.

Global distribution of microplastic contaminants in aquatic environments and their remediation strategies.

This review describes the occurrence and distribution of microplastics in freshwater and marine environments in recent years (2017-2022). Use of microplastics often results in contamination of aquatic environments, threatens biodiversity, and creates the need for environmental remediation. Such remediation strategies can involve primary, secondary, and tertiary treatments. Tertiary treatment is a frequent research subject due to its high efficiency and the possibility for advancements and enhancements. This study discusses tertiary treatments with remediation efficiencies of 95% and greater and their advantages, disadvantages, and future perspectives. Biochar-mediated remediation of microplastics is an effective method that may be able to achieve efficiencies approaching 100%. The study concludes by exploring methods of removing microplastics, including constructed wetlands and biochar, which offer high efficiency. PRACTITIONER POINTS: Tertiary treatments are an effective microplastic remediation strategy applicable succeeding secondary or primary treatments or as an individual remediation strategy. Biochar is a highly efficient adsorbent for microplastic remediation from aquatic environment with eco-friendly aspect and reusability. Modifications in tertiary treatments and enhancement in remediation efficiency are still a subject of research for future studies.

A rare case of congenital ranula.

Simple ranula is a cystic swelling confined to the floor of mouth while plunging ranula presents with extension to the neck. Congenital ranula presenting with symptoms of feeding difficulties is a very rare occurrence. There is paucity of literature regarding the management of large congenital ranula. Varied treatment options are described for the management of ranula with variable recurrence rate. In paediatric and adult patients, ranula is considered as a type of extravasation cyst and removal of sublingual gland is advocated to remove the sources of extravasation. Congenital ranula is usually a variant of retention cyst and should be treated with marsupialisation or simple excision of cyst while cyst excision with sublingual sialadenectomy should be reserved for recurrent cases.

Effect of Skeletal Paracrine Signals on the Proliferation of Interzone Cells.

OBJECTIVE: Articular cartilage in mammals has limited intrinsic capacity to repair structural defects, a fact that contributes to the chronic and progressive nature of osteoarthritis. In contrast, Mexican axolotl salamanders have demonstrated the remarkable ability to spontaneously and completely repair large joint cartilage lesions, a healing process that involves interzone cells in the intraarticular space. Furthermore, interzone tissue transplanted into skeletal defects in the axolotl salamander demonstrates a multi-differentiation potential. Cellular and molecular mechanisms of this repair process remain unclear. The objective of this study was to examine whether paracrine mitogenic signals are an important variable in the interaction between interzone cells and the skeletal microenvironment. DESIGN: The paracrine regulation of the proliferation of equine interzone cells was evaluated in an in vitro co-culture system. Cell viability and proliferation were measured in equine fetal interzone cells after exposure to conditioned medium from skeletal and nonskeletal primary cell lines. Steady-state expression was determined for genes encoding 37 putative mitogens secreted by cells that generated the conditioned medium. RESULTS: All experimental groups of conditioned media elicited a mitogenic response in interzone cells. Fetal anlage chondrocytes (P < 0.0001) and dermal fibroblasts (P < 0.0001) conditioned medium showed a significantly higher mitogenic potential compared with interzone cells. Conditioned medium from bone marrow-derived cells elicited a significantly higher proliferative response relative to that from young adult articular chondrocytes (P < 0.0001) or dermal fibroblasts (P < 0.0001). Sixteen genes had expression patterns consistent with the functional proliferation assays. CONCLUSIONS: The results indicate a mitogenic effect of skeletal paracrine signals on interzone cells.

An observation from an online survey: is fresh gas flow used for sevoflurane and desflurane different from isoflurane based anesthesia?

Minimal uses of fresh gas flow (FGF) during volatile inhalational agents based anesthesia are gaining popularity for many reasons. However, the practice pattern is not uniform. Even the same anesthesiologist uses different FGF for different agents. The present study was aimed to evaluate the variation in the practice pattern of FGF used in context to volatile agents used. With departmental approval, the present study was conducted by reviewing the data of a previously conducted cross-sectional survey. The survey was conducted from January 2018 to May 2018 using SurveyMonkey® . Anesthesiologists working in different organizations across India were approached through e-mail and WhatsApp and anonymous responses were collected. The responses which contained FGF data for isoflurane and for at least one of either sevoflurane and/or desflurane were included. A total of 236 eligible responses were analyzed. The FGFs used by different anesthesiologists were very much inconsistent; only 5.1% used FGF < 600 mL/min and 19.1% used 600-1000 mL/min consistently for all three agents. There was a significant variation of FGF used for sevoflurane and desflurane as compared to isoflurane. Use of FGF of < 1000 mL/min was significantly higher for the desflurane as compared to both isoflurane and sevoflurane. The uses of lower FGF greatly vary both at intrapersonal as well as interpersonal level. The possibility of using FGF < 1000 mL/min is significantly higher with desflurane as compared to isoflurane. Volatile anesthetic agent appears to be a factor for the decision making on the use of low flow anesthesia.

Advances in gene ontology utilization improve statistical power of annotation enrichment.

Gene-annotation enrichment is a common method for utilizing ontology-based annotations in gene and gene-product centric knowledgebases. Effective utilization of these annotations requires inferring semantic linkages by tracing paths through edges in the ontological graph, referred to as relations. However, some relations are semantically problematic with respect to scope, necessitating their omission or modification lest erroneous term mappings occur. To address these issues, we created the Gene Ontology Categorization Suite, or GOcats-a novel tool that organizes the Gene Ontology into subgraphs representing user-defined concepts, while ensuring that all appropriate relations are congruent with respect to scoping semantics. Here, we demonstrate the improvements in annotation enrichment by re-interpreting edges that would otherwise be omitted by traditional ancestor path-tracing methods. Specifically, we show that GOcats' unique handling of relations improves enrichment over conventional methods in the analysis of two different gene-expression datasets: a breast cancer microarray dataset and several horse cartilage development RNAseq datasets. With the breast cancer microarray dataset, we observed significant improvement (one-sided binomial test p-value = 1.86E-25) in 182 of 217 significantly enriched GO terms identified from the conventional path traversal method when GOcats' path traversal was used. We also found new significantly enriched terms using GOcats, whose biological relevancy has been experimentally demonstrated elsewhere. Likewise, on the horse RNAseq datasets, we observed a significant improvement in GO term enrichment when using GOcat's path traversal: one-sided binomial test p-values range from 1.32E-03 to 2.58E-44.

Synthesis and biological evaluation of potent benzoselenophene and heteroaromatic analogues of (S)-1-(chloromethyl)-8-methoxy-2,3-dihydro-1H-benzo[e]indol-5-ol (seco-MCBI).

A diverse series of compounds (18a-x) were synthesized from (S)-1-(chloromethyl)-8-methoxy-2,3-dihydro-1H-benzo[e]indol-5-ol (seco-MCBI) and benzoselenophene or heteroaromatic acids. These new compounds were evaluated for their cytotoxicity against the human gastric NCI-N87 and human ovarian SK-OV3 cancer cell lines. The incorporation of a methoxy substituent at the C-7 position of the seco-CBI unit enhances the cytotoxicity through its additional van der Waals interaction and gave a much higher potency than the corresponding seco-CBI-based analogues. Similarly, the seco-MCBI-benzoselenophene conjugates (18h-x) exhibited substitution effects on biological activity, and the N-butyramido and N-methylthiopropanamido analogues are highly potent, possessing >77- and >24-fold better activity than seco-MCBI-TMI for the SK-OV3 and NCI-N87 cell lines, respectively.

Emerging trends in molecular recognition: utility of weak aromatic interactions.

Aromatic interactions play a vital role in chemistry and biology. As about 20% are aromatic in nature, so the role of aromatic interactions become prominent in drug receptor interactions. Not only in drug receptor interactions but also in crystal engineering, protein folding, stacking interactions in DNA/RNA the role of the interactions is of utmost importance. With the emergence of supramolecular chemistry dendrimers, tweezers, rotaxanes, catenanes, and several supramolecular aggregates are associated with aromatic interactions. The mechanism of such interactions is still unknown by the replacement of a small substituent from the aromatic molecule may lead or destroy the interactions. In the present review several models are being discussed with arene interactions under selected heads.

Two Phosphoglucomutase Paralogs Facilitate Ionophore-Triggered Secretion of the Toxoplasma Micronemes.

Paralogs of the widely prevalent phosphoglucomutase (PGM) protein called parafusin function in calcium (Ca2+)-mediated exocytosis across eukaryotes. In Toxoplasma gondii, the parafusin-related protein 1 (PRP1) has been associated with Ca2+-dependent microneme organelle secretion required for essential processes like host cell invasion and egress. Using reverse genetics, we observed PRP1 to be dispensable for completion of the lytic cycle, including host cell invasion and egress by the parasite. However, the absence of the gene affected increased microneme release triggered by A23187, a Ca2+ ionophore used to raise the cytoplasmic Ca2+ concentration mimicking the physiological role of Ca2+ during invasion and egress. The basal levels of constitutive microneme release in extracellular parasites and phosphatidic acid-triggered microneme secretion were unaffected in the mutant. The phenotype of the deletion mutant of the second PGM-encoding gene in Toxoplasma, PGM2, was similar to the phenotype of the PRP1 deletion mutant. Furthermore, the ability of the tachyzoites to induce acute infection in the mice remained normal in the absence of both PGM paralogs. Our data thus reveal that the microneme secretion upon high Ca2+ flux is facilitated by the Toxoplasma PGM paralogs, PRP1 and PGM2. However, this protein-mediated release is neither essential for lytic cycle completion nor for acute virulence of the parasite. IMPORTANCE Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite's lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice.

Differential Roles for Inner Membrane Complex Proteins across Toxoplasma gondii and Sarcocystis neurona Development.

The inner membrane complex (IMC) of apicomplexan parasites contains a network of intermediate filament-like proteins. The 14 alveolin domain-containing IMC proteins in Toxoplasma gondii fall into different groups defined by their distinct spatiotemporal dynamics during the internal budding process of tachyzoites. Here, we analyzed representatives of different IMC protein groups across all stages of the Toxoplasma life cycle and during Sarcocystis neurona asexual development. We found that across asexually dividing Toxoplasma stages, IMC7 is present exclusively in the mother's cytoskeleton, whereas IMC1 and IMC3 are both present in mother and daughter cytoskeletons (IMC3 is strongly enriched in daughter buds). In developing macro- and microgametocytes, IMC1 and -3 are absent, whereas IMC7 is lost in early microgametocytes but retained in macrogametocytes until late in their development. We found no roles for IMC proteins during meiosis and sporoblast formation. However, we observed that IMC1 and IMC3, but not IMC7, are present in sporozoites. Although the spatiotemporal pattern of IMC15 and IMC3 suggests orthologous functions in Sarcocystis, IMC7 may have functionally diverged in Sarcocystis merozoites. To functionally characterize IMC proteins, we knocked out IMC7, -12, -14, and -15 in Toxoplasma. IMC14 and -15 appear to be involved in switching between endodyogeny and endopolygeny. In addition, IMC7, -12, and -14, which are all recruited to the cytoskeleton outside cytokinesis, are critical for the structural integrity of extracellular tachyzoites. Altogether, stage- and development-specific roles for IMC proteins can be discerned, suggesting different niches for each IMC protein across the entire life cycle. IMPORTANCE The inner membrane complex (IMC) is a defining feature of apicomplexan parasites key to both their motility and unique cell division. To provide further insights into the IMC, we analyzed the dynamics and functions of representative alveolin domain-containing IMC proteins across developmental stages. Our work shows universal but distinct roles for IMC1, -3, and -7 during Toxoplasma asexual division but more specialized functions for these proteins during gametogenesis. In addition, we find that IMC15 is involved in daughter formation in both Toxoplasma and Sarcocystis. IMC14 and IMC15 function in limiting the number of Toxoplasma offspring per division. Furthermore, IMC7, -12, and -14, which are recruited in the G1 cell cycle stage, are required for stress resistance of extracellular tachyzoites. Thus, although the roles of the different IMC proteins appear to overlap, stage- and development-specific behaviors indicate that their functions are uniquely tailored to each life stage requirement.

Biophysical characterization of in vitro bound Streptomyces peucetius daunorubicin-serine protease complex.

A serine protease of Streptomyces peucetius is found in association with daunorubicin in the culture filtrate and co-purifies as a complex as reported earlier by us (Dubey et al., 2013). The same protease was purified without drug attachment from dpsA(-) mutant of S. peucetius, which does not produce daunorubicin. Drug-protein complex was made in vitro by mixing daunorubicin and the protease. Spectral analysis and circular dichroism (CD) analysis were employed to determine the interaction between daunorubicin and the protease. Our study showed that interaction of daunorubicin with the protease affects the spectral characteristics of the drug and changes the secondary structure of the protein. Thin layer chromatography (TLC) analysis showed that the drug-protein interaction results in partial conversion of the drug to aglyconic form. The complex formation implies sequestration of the drug when it attains potentially lethal level in the extracellular milieu of S. peucetius culture.