Multiple roles for separase auto-cleavage during the G2/M transition

The cysteine protease separase triggers anaphase onset by cleaving chromosome-bound cohesin. In humans, separase also cleaves itself at multiple sites, but the biological significance of this reaction has been elusive. Here we show that preventing separase auto-cleavage, via targeted mutagenesis of the endogenous hSeparase locus in somatic cells, interferes with entry into and progression through mitosis. The initial delay in mitotic entry was not dependent on the G2 DNA damage checkpoint, but rather involved improper stabilization of the mitosis-inhibiting kinase Wee1. During M phase, cells deficient in separase auto-cleavage exhibited striking defects in spindle assembly and metaphase chromosome alignment, revealing an additional early mitotic function for separase. Both the G2 and M phase phenotypes could be recapitulated by separase RNA interference and corrected by re-expressing wild-type separase in trans. We conclude that separase auto-cleavage coordinates multiple aspects of the G2/M programme in human cells, thus contributing to the timing and efficiency of chromosome segregation.     

Cytodiagnosis of simple and proliferating trichilemmal cysts

OBJECTIVE: To elucidate the cytologic findings in simple trichilemmal (pilar) cysts and proliferating trichilemmal cysts (pilar tumors) and the clinical importance of these lesions. STUDY DESIGN: Aspirates from 12 simple pilar cysts and three pilar tumors, all histologically confirmed, were analyzed with a view to elaborating on specific cytologic features enabling a distinction from epidermal cysts and other adnexal tumors. RESULTS: Aspirates from pilar cysts showed two different pictures, depending on the age of the cyst. Young pilar cysts showed aspirates with an abundant background of blotchy keratin with or without calcification and inflammation. Older (degenerating) cysts showed oily fluid debris with cholesterol crystals and inflammatory cells. The epithelial component was sparse, and only an occasional syncitial cluster of small squamoid cells was seen. As opposed to this, epidermal cysts usually showed a cleaner background, with very cellular aspirates containing many nucleate and anucleate squames, keratin flakes, platelike crystals and no calcification. The pilar tumors showed a lesser amount of keratin as compared to pilar cysts and yielded large and small clusters of squamoid and basaloid cells, a few of which showed an abrupt association with anucleate, keratinized globules. CONCLUSION: The cytologic diagnosis of a pilar cyst should be made especially in scalp cysts, which yield either abundant, blotchy keratin or oily, cholesterol-rich debris with a sparse epithelial component and which lack a mixture of anucleate and nucleate squames. On aspiration, pilar tumors yield comparatively more cells and less keratin and show small, basaloid or squamoid cells abruptly associated with keratin globules. The cytologic diagnosis of pilar cysts is important because these cysts recur if incompletely excised and often undergo transformation to pilar tumors. Similarly, pilar tumors often clinically mimic squamous carcinomas, and hence there is a need to outline definitive cytologic features.     

Ultrasound‐assisted three‐phase partitioning of polyphenol oxidase from potato peel (Solanum tuberosum)

Conventional three phase partitioning (TPP) and ultrasound assisted three phase partitioning (UATPP) were optimized for achieving the maximum extraction and purification of polyphenol oxidase ( PPO) from waste potato peels. Different process parameters such as ammonium sulfate (NH₄)₂SO₄ concentration, crude extract to t‐butanol ratio, time, temperature and pH were studied for conventional TPP. Except agitation speed, the similar parameters were also optimized for UATPP. Further additional parameters were also studied for UATPP viz. irradiation time at different frequencies, duty cycle and, rated power in order to obtain the maximum purification factor and recovery of PPO. The optimized conditions for conventional TPP were (NH₄)₂SO₄ 0‐40% (w/v), extract to t‐butanol ratio 1:1 (v/v), time 40 min and pH 7 at 30°C. These conditions provided 6.3 purification factor and 70% recovery of PPO from bottom phase. On the other hand, UATPP gives maximum purification fold of 19.7 with 98.3% recovery under optimized parameters which includes (NH₄)₂SO₄ 0‐40% (w/v), crude extract to t‐butanol ratio 1: 1 (v/v) pH 7, irradiation time 5 min with 25 kHz, duty cycle 40% and rated power 150W at 30°C. UATPP delivers higher purification factor and % recovery of PPO along with reduced operation time from 40 min to 5 min when compared with TPP. SDS PAGE showed partial purification of PPO enzyme with UATPP with molecular weight in the range of 26‐36 kDa. Results reveal that UATPP would be an attractive option for the isolation and purification of PPO without need of multiple steps. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1340–1347, 2015     

Effectiveness Study of Paromomycin IM Injection (PMIM) for the Treatment of Visceral Leishmaniasis (VL) in Bangladesh

Background

This study was conducted in Bangladeshi patients in an outpatient setting to support registration of Paromomycin Intramuscular Injection (PMIM) as a low-cost treatment option in Bangladesh.

Methodology

This Phase IIIb, open-label, multi-center, single-arm trial assessed the efficacy and safety of PMIM administered at 11 mg/kg (paromomycin base) intramuscularly once daily for 21 consecutive days to children and adults with VL in a rural outpatient setting in Bangladesh. Patients ≥5 and ≤55 years were eligible if they had signs and symptoms of VL (intermittent fever, weight loss/decreased appetite, and enlarged spleen), positive rK39 test, and were living in VL-endemic areas. Compliance was the percentage of enrolled patients who received 21 daily injections over no more than 22 days. Efficacy was evaluated by initial clinical response, defined as resolution of fever and reduction of splenomegaly at end of treatment, and final clinical response, defined as the absence of new clinical signs and symptoms of VL 6 months after end of treatment. Safety was assessed by evaluation of adverse events.

Principal Findings

A total of 120 subjects (49% pediatric) were enrolled. Treatment compliance was 98.3%. Initial clinical response in the Intent-to-Treat population was 98.3%, and final clinical response 6 months after end of treatment was 94.2%. Of the 119 subjects who received ≥1 dose of PMIM, 28.6% reported at least one adverse event. Injection site pain was the most commonly reported adverse event. Reversible renal impairment and/or hearing loss were reported in 2 subjects.

Conclusions/Significance

PMIM was an effective and safe treatment for VL in Bangladesh. The short treatment duration and lower cost of PMIM compared with other treatment options may make this drug a preferred treatment to be investigated as part of a combination therapy regimen. This study supports the registration of PMIM for use in government health facilities in Bangladesh.

Trial Registration

ClinicalTrials.gov identifier: NCT01328457     

New target for inhibition of bacterial RNA polymerase: 'switch region'

A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery.     

Cellular magnetic resonance imaging of monocyte-derived dendritic cell migration from healthy donors and cancer patients as assessed in a scid mouse model

BACKGROUND AIMS. The use of dendritic cells (DC) as an adjuvant in cell-based immunotherapeutic cancer vaccines is a growing field of interest. A reliable and non-invasive method to track the fate of autologous DC following their administration to patients is required in order to confirm that clinically sufficient numbers are reaching the lymph node (LN). We demonstrate that an immunocompromised mouse model can be used to conduct translational studies employing cellular magnetic resonance imaging (MRI). Such studies can provide clinically relevant information regarding the migration potential of clinical-grade DC used in cancer immunotherapies. METHODS. Human monocyte-derived dendritic cells (mo-DC) were generated from negatively selected monocytes obtained from either healthy donors or cancer patients. DC were labeled with superparamagnetic iron oxide (SPIO) nanoparticles in order to track them in vivo in a CB17scid mouse model using cellular MRI. SPIO did not have any adverse effects on DC phenotype or function, independent of donor type. Cellular MRI readily detected migration of SPIO-loaded DC in CB17scid mice. No differences in migration were observed between DC obtained from healthy donors and those obtained from donors undergoing autologous stem cell transplant for cancer therapy. CONCLUSIONS. Cellular MRI provided semi-quantitative image data that corresponded with data obtained by digital morphometry, validating cellular MRI's potential to assess DC migration in DC-based cancer immunotherapy clinical trials.     

A novel disulfide-rich protein motif from avian eggshell membranes

Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X(4)-C-X(5)-C-X(8)-C-X(6) pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata) and in the oviparous green anole lizard (Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM fibers.     

Optimized protocol for efficient transfection of dendritic cells without cell maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection¹. Detection of pathogenic antigen by naïve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response^2,3. Thus, DCs link the innate and adaptive immune systems. The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs⁴.Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)^5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs^9,10,11,12. Electroporation has been used with mixed results^13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation¹⁶. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFNβ. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor^16,17, at both the mRNA and protein levels. Download video file.^{(52M, mov)}     

Effects of hesperidin,a flavanone glycoside interaction on the conformation,stability, and aggregation of lysozyme: multispectroscopic and molecular dynamic simulation studies?

Hesperidin (HESP), a flavanone glycoside, shows high antioxidant properties and posses ability to go through the blood–brain barrier. Therefore, it could be a potential drug molecule against aggregation based diseases such as Alzheimer’s, Parkinson’s, and systemic amyloidoses. In this work, we investigated the potential of HESP to interact with hen egg-white lysozyme (HEWL) monomer and prevent its aggregation. The HESP–HEWL binding studies were performed using a fluorescence quenching technique, molecular docking and molecular dynamics simulations. We found a strong interaction of HESP with the lysozyme monomer (K_a, ~ 5 × 10⁴ M^?1) mainly through hydrogen bonding, water bridges, and hydrophobic interactions. We showed that HESP molecule spanned the highly aggregation prone region (amino acid residues 48-101) of HEWL and prevented its fibrillar aggregation. Further, we found that HESP binding completely inhibited amorphous aggregation of the protein induced by disulfide-reducing agent tries-(2-carboxyethyl) phosphine. Conformational and stability studies as followed by various tertiary and secondary structure probes revealed that HESP binding only marginally affected the lysozyme monomer conformation and increased both stability and reversibility of the protein against thermal denaturation. Future studies should investigate detail effects of HESP on solvent dynamics, structure, and toxicity of various aggregates. The answers to these questions will not only target the basic sciences, but also have application in biomedical and biotechnological sciences.