Aggregation prone regions in human proteome: Insights from large‐scale data analyses

Protein aggregation leads to several burdensome human maladies, but a molecular level understanding of how human proteome has tackled the threat of aggregation is currently lacking. In this work, we survey the human proteome for incidence of aggregation prone regions (APRs), by using sequences of experimentally validated amyloid‐fibril forming peptides and via computational predictions. While approximately 30 human proteins are currently known to be amyloidogenic, we found that 260 proteins (～1% of human proteome) contain at least one experimentally validated amyloid‐fibril forming segment. Computer predictions suggest that more than 80% of the human proteins contain at least one potential APR and approximately two‐thirds (65%) contain two or more APRs; spanning 3–5% of their sequences. Sequence randomizations show that this apparently high incidence of APRs has been actually significantly reduced by unique amino acid composition and sequence patterning of human proteins. The human proteome has utilized a wide repertoire of sequence‐structural optimization strategies, most of them already known, to minimize deleterious consequences due to the presence of APRs while simultaneously taking advantage of their order promoting properties. This survey also found that APRs tend to be located near the active and ligand binding sites in human proteins, but not near the post translational modification sites. The APRs in human proteins are also preferentially found at heterotypic interfaces rather than homotypic ones. Interestingly, this survey reveals that APRs play multiple, often opposing, roles in the human protein sequence‐structure‐function relationships. Insights gained from this work have several interesting implications towards novel drug discovery and development. Proteins 2017; 85:1099–1118. © 2017 Wiley Periodicals, Inc.     

mGluR long‐term depression regulates GluA2 association with COPII vesicles and exit from the endoplasmic reticulum

mGluR long‐term depression (mGluR‐LTD) is a form of synaptic plasticity induced at excitatory synapses by metabotropic glutamate receptors (mGluRs). mGluR‐LTD reduces synaptic strength and is relevant to learning and memory, autism, and sensitization to cocaine; however, the mechanism is not known. Here we show that activation of Group I mGluRs in medium spiny neurons induces trafficking of GluA2 from the endoplasmic reticulum (ER) to the synapse by enhancing GluA2 binding to essential COPII vesicle proteins, Sec23 and Sec13. GluA2 exit from the ER further depends on IP3 and Ryanodine receptor‐controlled Ca²⁺ release as well as active translation. Synaptic insertion of GluA2 is coupled to removal of high‐conducting Ca²⁺‐permeable AMPA receptors from synapses, resulting in synaptic depression. This work demonstrates a novel mechanism in which mGluR signals release AMPA receptors rapidly from the ER and couple ER release to GluA2 synaptic insertion and GluA1 removal.     

High-throughput screening-based identification of paramyxovirus inhibitors

Several members of the paramyxovirus family constitute major human pathogens that, collectively, are responsible for major morbidity and mortality worldwide. In an effort to develop novel therapeutics against measles virus (MV), a prominent member of the paramyxovirus family, the authors report a high-throughput screening protocol that uses a nonrecombinant primary MV strain as targets. Implementation of the assay has yielded 60 hit candidates from a 137,500-entry library. Counterscreening and generation of dose-response curves narrows this pool to 35 compounds with active concentrations 500. Library mining for structural analogs of several confirmed hits combined with retesting of identified candidates reveals a high accuracy of primary hit identification. Eleven of the confirmed hits interfere with viral entry, whereas the remaining 24 compounds target postentry steps of the viral life cycle. Activity testing against selected members of the paramyxovirus family reveals 3 patterns of activity: 1) exclusively MV-specific blockers, 2) inhibitors of MV and related viruses of the same genus, and 3) broader range inhibitors with activity against a different Paramyxovirinae genus. Representatives of the last class may open avenues for the development of broad-range paramyxovirus inhibitors through hit-to-lead chemistry. ( Journal of Biomolecular Screening 2008:591-608).     

Chromosome 7 and 19 Trisomy in Cultured Human Neural Progenitor Cells

Background

Stem cell expansion and differentiation is the foundation of emerging cell therapy technologies. The potential applications of human neural progenitor cells (hNPCs) are wide ranging, but a normal cytogenetic profile is important to avoid the risk of tumor formation in clinical trials. FDA approved clinical trials are being planned and conducted for hNPC transplantation into the brain or spinal cord for various neurodegenerative disorders. Although human embryonic stem cells (hESCs) are known to show recurrent chromosomal abnormalities involving 12 and 17, no studies have revealed chromosomal abnormalities in cultured hNPCs. Therefore, we investigated frequently occurring chromosomal abnormalities in 21 independent fetal-derived hNPC lines and the possible mechanisms triggering such aberrations.

Methods and Findings

While most hNPC lines were karyotypically normal, G-band karyotyping and fluorescent in situ hybridization (FISH) analyses revealed the emergence of trisomy 7 (hNPC⁺⁷) and trisomy 19 (hNPC⁺¹⁹), in 24% and 5% of the lines, respectively. Once detected, subsequent passaging revealed emerging dominance of trisomy hNPCs. DNA microarray and immunoblotting analyses demonstrate epidermal growth factor receptor (EGFR) overexpression in hNPC⁺⁷ and hNPC⁺¹⁹ cells. We observed greater levels of telomerase (hTERT), increased proliferation (Ki67), survival (TUNEL), and neurogenesis (β_III-tubulin) in hNPC⁺⁷ and hNPC⁺¹⁹, using respective immunocytochemical markers. However, the trisomy lines underwent replicative senescence after 50–60 population doublings and never showed neoplastic changes. Although hNPC⁺⁷ and hNPC⁺¹⁹ survived better after xenotransplantation into the rat striatum, they did not form malignant tumors. Finally, EGF deprivation triggered a selection of trisomy 7 cells in a diploid hNPC line.

Conclusions

We report that hNPCs are susceptible to accumulation of chromosome 7 and 19 trisomy in long-term cell culture. These results suggest that micro-environmental cues are powerful factors in the selection of specific hNPC aneuploidies, with trisomy of chromosome 7 being the most common. Given that a number of stem cell based clinical trials are being conducted or planned in USA and a recent report in PLoS Medicine showing the dangers of grafting an inordinate number of cells, these data substantiate the need for careful cytogenetic evaluation of hNPCs (fetal or hESC-derived) before their use in clinical or basic science applications.     

Role of Calmodulin-Calmodulin Kinase II,cAMP/Protein Kinase A and ERK 1/2 on Aeromonas hydrophila-Induced Apoptosis of Head Kidney Macrophages

The role of calcium (Ca²⁺) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca²⁺-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca²⁺ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.     

Caprylate-Conjugated Cisplatin for the Development of Novel Liposomal Formulation

Cisplatin, first (platinum) compound to be evolved as an anticancer agent, has found its important place in cancer chemotherapy. However, the dose-dependent toxicities of cisplatin, namely nephrotoxicity, ototoxicity, peripheral neuropathy, and gastrointestinal toxicity hinder its widespread use. Liposomes can reduce the toxicity of cisplatin and provide a better therapeutic action, but the low lipid solubility of cisplatin hinders its high entrapment in such lipid carrier. In the present investigation, positively charged reactive aquated species of cisplatin were complexed with negatively charged caprylate ligands, resulting in enhanced interaction of cisplatin with lipid bilayer of liposomes and increase in its encapsulation in liposomal carrier. Prepared cisplatin liposomes were found to have a vesicular size of 107.9 ± 6.2 nm and zeta potential of −3.99 ± 3.45 mV. The optimized liposomal formulation had an encapsulation efficiency of 96.03 ± 1.24% with unprecedented drug loading (0.21 mg cisplatin / mg of lipids). The in vitro release studies exhibited a pH-dependent release of cisplatin from liposomes with highest release (67.55 ± 3.65%) at pH 5.5 indicating that a maximum release would occur inside cancer cells at endolysosomal pH. The prepared liposomes were found to be stable in the serum and showed a low hemolytic potential. In vitro cytotoxicity of cisplatin liposomes on A549 lung cancer cell line was comparable to that of cisplatin solution. The developed formulation also had a significantly higher median lethal dose (LD₅₀) of 23.79 mg/kg than that of the cisplatin solution (12 mg/kg). A promising liposomal formulation of cisplatin has been proposed that can overcome the disadvantages associated with conventional cisplatin therapy and provide a higher safety profile.Key Words: cisplatin, complexation, cytotoxicity, LD₅₀, liposome     

The Asparaginyl Endopeptidase Legumain Is Essential for Functional Recovery after Spinal Cord Injury in Adult Zebrafish

Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.     

Does Video Gaming Affect Orthopaedic Skills Acquisition? A Prospective Cohort-Study

Introduction

Previous studies have suggested that there is a positive correlation between the extent of video gaming and efficiency of surgical skill acquisition on laparoscopic and endovascular surgical simulators amongst trainees. However, the link between video gaming and orthopaedic trauma simulation remains unexamined, in particular dynamic hip screw (DHS) stimulation.

Objective

To assess effect of prior video gaming experience on virtual-reality (VR) haptic-enabled DHS simulator performance.

Methods

38 medical students, naïve to VR surgical simulation, were recruited and stratified relative to their video gaming exposure. Group 1 (n = 19, video-gamers) were defined as those who play more than one hour per day in the last calendar year. Group 2 (n = 19, non-gamers) were defined as those who play video games less than one hour per calendar year. Both cohorts performed five attempts on completing a VR DHS procedure and repeated the task after a week. Metrics assessed included time taken for task, simulated flouroscopy time and screw position. Median and Bonett-Price 95% confidence intervals were calculated for seven real-time objective performance metrics. Data was confirmed as non-parametric by the Kolmogorov-Smirnov test. Analysis was performed using the Mann-Whitney U test for independent data whilst the Wilcoxon signed ranked test was used for paired data. A result was deemed significant when a two-tailed p-value was less than 0.05.

Results

All 38 subjects completed the study. The groups were not significantly different at baseline. After ten attempts, there was no difference between Group 1 and Group 2 in any of the metrics tested. These included time taken for task, simulated fluoroscopy time, number of retries, tip-apex distance, percentage cut-out and global score.

Conclusion

Contrary to previous literature findings, there was no correlation between video gaming experience and gaining competency on a VR DHS simulator.     

Systematic status of true katydids Sathrophyllia (Orthoptera,Tettigonioidea, Pseudophyllinae) from Pakistan,with description of two new species

The genus Sathrophyllia Stål, 1874 from Pakistan is reviewed with four species recorded. The diagnostic characters are given and two new species Sathrophyllia saeedi sp. n. and Sathrophyllia irshadi sp. n. are described. In addition to that Sathrophyllia nr. rugosa (Linnaeus, 1758) and Sathrophyllia femorata (Fabricius, 1787) are re-described. Further information on the distribution and ecology of the species is given and a key to studied species of Sathrophyllia is presented. Sathrophyllia femorata (Fabricius, 1787) and Sathrophyllia rugosa (Linnaeus, 1758) are recorded from Rawalakot (KPK) and Tharparker (Sindh), Pakistan for first the time.