Inhibition of huntingtin aggregation by its N-terminal 17-residue peptide and its analogs.

The N-terminal 17-residue stretch of huntingtin (httN17) folds into an amphipathic α-helix. The httN17-harboring polyQ peptides form oligomers that are mediated via the assembly of the httN17 α-helices. The oligomerization results in higher local concentration of the polyglutamine (polyQ) region, thereby facilitating amyloid formation. The httN17 co-assembles with the httN17-harbouring polyQ peptides, thereby reducing the local polyQ concentration, and consequently inhibiting aggregation. This study presents the aggregation inhibition of the exon I region of pathogenic huntingtin by httN17 and its analogs. The C-terminal amidation of httN17 is found to be essential for activity. The httN17 peptides with free amino terminus and the acetylated amino terminus possess comparable activity. The httN17 analog, wherein the Leu7 and Ala10 are substituted with 2-aminoisobutyric acid residues, exhibits significantly higher activity than the native httN17.

Interaction of cavin-1/PTRF leucine zipper domain 2 and its congenital generalized lipodystrophy mutant with model membranes.

The organization of caveolae ultrastructures in the plasma membrane and the functions they dictate are mediated by membrane-embedded caveolins (caveolin-1, 2, 3) and peripherally attached cavins (cavin-1, 2, 3, 4). Mutations in caveolin and cavin genes are associated with a variety of human diseases. Cavin-1/PTRF mutations are known to contribute to several human pathologies, including muscular dystrophy and congenital generalized lipodystrophy (CGL). In the present study, we investigated the membrane interaction of the second leucine zipper domain (LZD2) of cavin-1 and the analogous peptide stretch in its CGL frameshift mutant (p.Glu176Argfs). The fluorescence data from the Trp-tagged peptides suggest binding of both wild-type and mutant peptide with negatively-charged membranes. The mutant peptide displayed a rather enhanced interaction compared to the wild-type peptide. In addition, the mutant peptide displayed appreciable binding to the lipid raft-mimicking cholesterol/sphingomyelin-rich vesicles as well. The alteration in the dynamics of peptide-lipid interaction is attributed to increased charge and hydrophilicity of the mutant peptides. Overall, these results suggest that the frameshift mutation in cavin-1/PTRF (p.Glu176Argfs) imparts high membrane-binding propensity to the region corresponding to LZD2, which is hitherto unknown to interact with membranes. Such interaction in the disease condition, in turn, could either alter the native membrane interaction dynamics of cavin-1/PTRF or possibly result in interaction with non-target membranes.

Modelling past and future peatland carbon dynamics across the pan-Arctic.

The majority of northern peatlands were initiated during the Holocene. Owing to their mass imbalance, they have sequestered huge amounts of carbon in terrestrial ecosystems. Although recent syntheses have filled some knowledge gaps, the extent and remoteness of many peatlands pose challenges to developing reliable regional carbon accumulation estimates from observations. In this work, we employed an individual- and patch-based dynamic global vegetation model (LPJ-GUESS) with peatland and permafrost functionality to quantify long-term carbon accumulation rates in northern peatlands and to assess the effects of historical and projected future climate change on peatland carbon balance. We combined published datasets of peat basal age to form an up-to-date peat inception surface for the pan-Arctic region which we then used to constrain the model. We divided our analysis into two parts, with a focus both on the carbon accumulation changes detected within the observed peatland boundary and at pan-Arctic scale under two contrasting warming scenarios (representative concentration pathway-RCP8.5 and RCP2.6). We found that peatlands continue to act as carbon sinks under both warming scenarios, but their sink capacity will be substantially reduced under the high-warming (RCP8.5) scenario after 2050. Areas where peat production was initially hampered by permafrost and low productivity were found to accumulate more carbon because of the initial warming and moisture-rich environment due to permafrost thaw, higher precipitation and elevated CO2 levels. On the other hand, we project that areas which will experience reduced precipitation rates and those without permafrost will lose more carbon in the near future, particularly peatlands located in the European region and between 45 and 55°N latitude. Overall, we found that rapid global warming could reduce the carbon sink capacity of the northern peatlands in the coming decades.

Amyloids and their untapped potential as hydrogelators.

Amyloid fibrils are cross-ß-sheet-rich fibrous aggregates. They were originally identified as disease-associated protein/peptide deposits. The cross-ß motif was consequently labelled as an alien and pathogenic fold. Subsequent research revealed that the fibrillar aggregates were benign, and the cytotoxicity in the amyloid diseases was attributed to the pre-fibrillar structures. Research in the past two decades has identified the native functional amyloids in organisms ranging from bacteria to human. The amyloid-like fibrils, therefore, are not necessarily pathogenic, and the cross-ß motif is very much native. This premise makes way for the amyloids to be used as biocompatible materials. Many naturally occurring amyloidogenic proteins/peptides or their fragments have been reported in the literature to form hydrogels. Hydrogels constitute one of the most interesting classes of soft materials that find application in diverse fields such as environmental, electronic, and biomedical engineering. Applications of hydrogels in medicine are particularly extensive. Among various classes of peptides that form hydrogels, the potential of amyloids is largely untapped. In this review, we have attempted to compile the literature on amyloid hydrogels and discuss their potential applications.

Aromatic-rich C-terminal region of LCI is a potent antimicrobial peptide in itself.

LCI is a 47-residue antimicrobial peptide produced by Bacillus subtilis. The peptide displays potent activity against plant pathogens, Xanthomonas and Pseudomonas. The peptide takes a compact 3-dimensional structure characterized by a four-stranded ß-sheet. The peptide is unusually rich in aromatic residues; 10 of the 47 residues are aromatic and 8 of them lie in the C-terminal region, LCI22-47. Here we report the antimicrobial activity of this C-terminal region against Gram-positive and Gram-negative bacteria. The C-terminal-amidated peptide displays potent activity against E. coli, methicillin and gentamicin-resistant S. aureus, and Xanthomonas oryzae pv. oryzae with lethal concentrations ≤4 µM. Membrane-binding assays indicate preferential binding to the negatively-charged lipids. The peptide permeabilizes the outer-membrane of E. coli indicating membrane-permeabilization as one of the mechanisms of killing. Interestingly, however, no inner-membrane permeabilization was observed, indicating that the membrane-permeabilization may not be the sole mechanism of action.

Limpid hydrogels from ß-turn motif-connected tandem repeats of Aß16-22.

Self-assembling peptides constitute an important class of functional biomaterials. A number of short amyloidogenic stretches have been identified from amyloid proteins. Such peptides, as such or through subtle modifications, can turn out to be promising candidates for functional biomaterials. End-capped Aß16-22, the well-studied amyloidogenic stretch from ß-amyloid, is reported to be non-hydrogelating up to 20 mM concentration. Here we investigated the hydrogelation propensity of Aß16-22 repeats connected through ß-turn-supporting motifs. The peptide repeats connected through Asn-Gly, Aib-DPro, and DPro-Gly formed transparent hydrogels at concentrations ≥2 mM. The repeats of the aromatic analog Aß16-22(F20Y) also resulted in similar hydrogels. Like other peptide-based gels reported earlier, these gels could trap the anticancer drug doxorubicin and displayed steady release in water. In addition, the gels supported the growth of mammalian cell lines, HEK-293 and RIN-5F. These data show that turn-inducing motifs can have marked effects on the hydrogelating propensity of self-assembling peptides.

Glycoprotein D peptide-based diagnostic approach for the detection of avian infectious laryngotracheitis antibodies.

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens, pheasants, and peafowl. It is caused by the alpha herpesvirus, infectious laryngotracheitis virus (ILTV). Glycoprotein D (gD) of ILTV is immunogenic and helps in its binding to the susceptible host cell receptor. In the present study, a recombinant gD protein was expressed in a prokaryotic system to develop a single serum dilution ELISA. In addition, two immunogenic peptides, corresponding to regions 77-89 and 317-328, were identified in gD protein. The peptides were synthesized using solid-phase peptide synthesis, purified using reversed-phase HPLC, and characterized using mass spectrometry. The peptides displayed a good titre and were found to be promising antigens to coat the ELISA plate to detect the ILTV antibodies in the serum sample. The developed ELISA showed 96.9% sensitivity, 87.5% specificity, and 95.3% accuracy as compared to OIE referenced standard indirect ILTV ELISA (whole viral coated). The assay may not differentiate vaccinated from infected birds when the flocks are administered with live attenuated vaccines. However, the assay could be useful to detect the disease condition in birds vaccinated with recombinant vaccine expressing glycoproteins other than gD. The developed ILTV single serum dilution ELISA could be an alternative to the existing diagnostics for the detection of ILTV antibodies.

Interaction of MreB-derived antimicrobial peptides with membranes.

Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB1-9 and its N-terminal acetylated analog, Ac-WMreB1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them.

Physiological acclimation dampens initial effects of elevated temperature and atmospheric CO2 concentration in mature boreal Norway spruce.

Physiological processes of terrestrial plants regulate the land-atmosphere exchange of carbon, water, and energy, yet few studies have explored the acclimation responses of mature boreal conifer trees to climate change. Here we explored the acclimation responses of photosynthesis, respiration, and stomatal conductance to elevated temperature and/or CO2 concentration ([CO2 ]) in a 3-year field experiment with mature boreal Norway spruce. We found that elevated [CO2 ] decreased photosynthetic carboxylation capacity (-23% at 25 °C) and increased shoot respiration (+64% at 15 °C), while warming had no significant effects. Shoot respiration, but not photosynthetic capacity, exhibited seasonal acclimation. Stomatal conductance at light saturation and a vapour pressure deficit of 1 kPa was unaffected by elevated [CO2 ] but significantly decreased (-27%) by warming, and the ratio of intercellular to ambient [CO2 ] was enhanced (+17%) by elevated [CO2 ] and decreased (-12%) by warming. Many of these responses differ from those typically observed in temperate tree species. Our results show that long-term physiological acclimation dampens the initial stimulation of plant net carbon assimilation to elevated [CO2 ], and of plant water use to warming. Models that do not account for these responses may thus overestimate the impacts of climate change on future boreal vegetation-atmosphere interactions.

Effect of tacticity-derived topological constraints in bactericidal peptides.

Topology is a key element in structure-activity relationship estimation while designing physiologically-active molecular constructs. Peptides may be a preferred choice for therapeutics, principally due to their biocompatibility, low toxicity and predictable metabolism. Peptide design only guarantees functional group constitution by opting specific amino acid sequence, and not their spatial orientation to bind and incite physiological response on chosen targets. This is principally because peptide conformation is subject to external flux, due to the isotactic stereochemistry of the peptide chain. Stereochemical engineering of the peptide main chain offers the possibility of multiplying the structural space of a typical sequence to many orders of magnitude, and limiting the otherwise fluxional non-specific functional group dispensation in space by offering greater conformational rigidity. We put to test, this conceptual possibility already established in theoretical models, by designing amphipathic peptide systems and experimenting with them on Gram-positive, Gram-negative and antibiotic-resistant bacteria. The unusual conformational rigidity and stability of syndiotactic peptides enable them to retain the designed electrostatic environment, while they encounter the membrane surface. All the six designed systems exhibited bactericidal activity, pointing to the utility and specificity of stereo-engineered peptide systems for therapeutic applications. Overall, we hope that this work provides important insights and useful directives in designing novel peptide systems with antimicrobial activity, by expanding the design space, incorporating D-amino acid as an additional design variable.

Alpha-synuclein and RNA viruses: Exploring the neuronal nexus.

Alpha-synuclein (α-syn), known for its pivotal role in Parkinson's disease, has recently emerged as a significant player in neurotropic RNA virus infections. Upregulation of α-syn in various viral infections has been found to impact neuroprotective functions by regulating neurotransmitter synthesis, vesicle trafficking, and synaptic vesicle recycling. This review focuses on the multifaceted role of α-syn in controlling viral replication by modulating chemoattractant properties towards microglial cells, virus-induced ER stress signaling, anti-oxidative proteins expression. Furthermore, the text underlines the α-syn-mediated regulation of interferon-stimulated genes. The review may help suggest potential therapeutic avenues for mitigating the impact of RNA viruses on the central nervous system by exploiting α-syn neuroprotective biology.

Peripheral blood quantitation of CD26 positive leukemic stem cells as a predictor of tyrosine kinase inhibitor response in chronic myeloid leukemia.

INTRODUCTION: Leukemic stem cells (LSCs) are the transcriptionally low/silent cells which are resistant to the tyrosine kinase inhibitor. These have been found to play a pivotal role in disease relapse in chronic myeloid leukemia (CML) cases. The present study evaluated the correlation of absolute CML-LSC count in the peripheral blood (PB) at diagnosis and achievement of major molecular response (MMR) at 12 months in patients of CML-CP. METHODS: This was a prospective, observational, non-interventional single center study including newly diagnosed adult (>18 yrs) CML-CP patients. Absolute CD26 + CML-LSC quantification was done by multiparametric flow cytometry. Patients were treated with Imatinib treatment and subsequently monitored at 3-month intervals for BCR::ABL transcript levels. MMR was defined as a BCR::ABL1 transcript level of less than 0.1% on international scale. RESULTS: A total of 89 patients were enrolled in the study out of which 40.5% achieved MMR at 12 months. There was a significant difference in the median absolute CML-LSC count of the patients who achieved MMR at 12 months as compared to those who did not (58.5 vs 368.1 cells/µL; p value <0.001). Using a ROC analysis, a count of <165.69 CML LSC/µL was identified to have a sensitivity of 83.8% and specificity of 72.4%, in predicting the MMR at 12 months. CONCLUSION: Absolute CML-LSC count at diagnosis in the PB predicts the MMR achievement at 12 months. An absolute count of less than 165 cells/µL is highly predictive of achieving MMR at 12 months.

Tau fibrillogenesis.

The protein tau is the most abundant microtubule associated protein in the central and peripheral nervous system. In the brain, tau plays a role in the assembly and stabilization of microtubules. The function of tau, however, appears to overlap with other microtubule binding proteins. The observation that tau is associated with neurodegenerative diseases has renewed interest in this protein. Various aspects of structure and biochemistry of tau, fibril formation and clinical perspectives, including therapeutic strategies are reviewed in this chapter.

Interfacial properties of α-synuclein's Parkinsonian variants.

Human alpha-synuclein (αS) is associated with the occurrence of Parkinson's disease. In the past decade, six autosomally dominant mutations have been identified in αS (SNCA) gene that translate into A30P, E46K, H50Q, G51D, A53E, and A53T mutations in the protein. These mutations alter the electrostatics and hydrophobicity of a cardinal region of the protein. A comprehensive comparison of interfacial properties of these Parkinsonian αS variants is crucial to understand their membrane dynamics. Here, we investigated the interfacial activity of these αS variants at air-aqueous interface. All the αS variants were found to possess comparable surface activity of ∼20-22 mN/m. Compression/expansion isotherms reveal a very distinct behaviour of the A30P variant compared to others. The Blodgett-deposited films were analysed using CD and LD spectroscopy as well as the atomic force microscopy. All the variants adopted predominantly α-helical conformation in these films. Atomic force microscopy of the Langmuir-Blodgett films revealed self-assembly at the interface. The lipid-penetration activity was also investigated using zwitterionic and negatively charged lipid monolayers.

Hexafluoroisopropanol induces self-assembly of ß-amyloid peptides into highly ordered nanostructures.

Deposition of insoluble fibrillar aggregates of ß-amyloid (Aß) peptides in the brain is a hallmark of Alzheimer's disease. Apart from forming fibrils, these peptides also exist as soluble aggregates. Fibrillar and a variety of nonfibrillar aggregates of Aß have also been obtained in vitro. Hexafluoroisopropanol (HFIP) has been widely used to dissolve Aß and other amyloidogenic peptides. In this study, we show that the dissolution of Aß40, 42, and 43 in HFIP followed by drying results in highly ordered aggregates. Although α-helical conformation is observed, it is not stable for prolonged periods. Drying after prolonged incubation of Aß40, 42, and 43 peptides in HFIP leads to structural transition from α-helical to ß-conformation. The peptides form short fibrous aggregates that further assemble giving rise to highly ordered ring-like structures. Aß16-22, a highly amyloidogenic peptide stretch from Aß, also formed very similar rings when dissolved in HFIP and dried. HFIP could not induce α-helical conformation in Aß16-22, and rings were obtained from freshly dissolved peptide. The rings formed by Aß40, 42, 43, and Aß16-22 are composed of the peptides in ß-conformation and cause enhancement in thioflavin T fluorescence, suggesting that the molecular architecture of these structures is amyloid-like. Our results clearly indicate that dissolution of Aß40, 42 and 43 and the amyloidogenic fragment Aß16-22 in HFIP results in the formation of annular amyloid-like structures.

Management of Maxillofacial Trauma in Road Traffic Accident (RTA) at Tertiary Care Center.

Road traffic accidents (RTA) are the major cause of maxillofacial injuries (MFIs) in developing countries (Akama et al. 2007). Road traffic accidents were reported to be the 9th most common cause of death and morbidity in the world and are expected to rise to 3rd position by 2020 (Peden et al. 2002). Maxillofacial injuries remain a serious clinical problem because of the involvement of complex anatomic region. Facial fractures occur most commonly in males in the third decade of life (Motamedi et al. 2014). The goal of treatment in facial fractures is to achieve anatomic reduction and restore function while increasing patient comfort and making postoperative care easier (Lachner et al. 1991). The aim of the study was to evaluate the cases of Maxillofacial injuries with the existing literature on its different presentation and management. An observational study was done from the patients of RTA with Maxillofacial injuries in ENT department and trauma centre of Sir T General hospital and Government Medical College, Bhavnagar for a period of 2 year. A total of 315 patients were included. Males are more commonly affected than females. The main etiological factor for RTA was motorcycle accidents. The trend of MFIs especially due to MCAs was on the rise after the age of 20 year. Anatomically the lower 1/3 section of the face was the most affected. Mandibular fractures were most common isolated fracture in MFIs. Open reduction and internal fixation (ORIF) by plating and screw was the treatment of choice for displaced, comminuted and multiple fractures of face. Facial trauma remains a major source of injury in all parts of the world. Its management involves many disciplines in the hospital setting, but knowledge of occlusion, the masticatory apparatus and anatomy is important for the best outcomes. This study was an analysis of demographic variables and outcome of the management adopted in patients presented to our department.

Impact on the replacement of Phe by Trp in a short fragment of Aß amyloid peptide on the formation of fibrils.

Aß(16-22) (Ac-KLVFFAE-NH(2) ) is one of the shortest amyloid fibril-forming sequences identified in ß-amyloid peptide. At neutral pH, the peptide forms fibrils in the concentration range of 0.2-2.0 mM after ≥ 10 days of incubation. Structures of the fibrils proposed based on solid-state NMR and MD simulations studies suggest antiparallel arrangement of ß-strands and aromatic interactions between the Phe residues. In an effort to examine the role of aromatic interactions between two Phe residues in Aß(16-22) , we have studied the self-assembly of Aß(16-22) (AßFF) and two of its variants, Ac-KLVFWAE-NH(2) (AßFW) and Ac-KLVWFAE-NH(2) (AßWF). The peptides were dissolved in methanol (MeOH) at a concentration of 1 mM and in water (AßFW and AßWF, 1 mM; AßFF, 330 µM). Peptide solutions (100 µM) were prepared in 50 mM sodium phosphate buffer at pH 7 by diluting from MeOH and water stock solutions. AßFW forms amyloid-like fibrils immediately from MeOH, as indicated by atomic force microscopy. Dilution of AßFW into phosphate buffer from stock solution prepared in MeOH results in fibrils, but with different morphology and dimensions. The secondary structure potentiated by MeOH seems to be important for the self-assembly of AßFW, as fibrils are not formed from water where the peptide is unordered. On the other hand, AßFF and AßWF do not form amyloid fibrils rapidly from any of the solvents used for dissolution. However, drying of AßWF from MeOH on mica surface gives rod-like and fibrous structures. Our study indicates that positioning of the aromatic residues F and W has an important role to play in promoting self-assembly of the Aß(16-22) peptides.

N-terminal acetylation does not alter α-synuclein's interfacial properties.

Alpha-synuclein (αS) is a membrane-binding protein found predominantly in neurons and erythrocytes. The protein remains unordered in aqueous solutions but folds into an α-helical structure when bound to membranes. Besides, it gets deposited as ß-sheet rich aggregates in diseases known as synucleinopathies. The native αS has been reported to be acetylated at the N-terminus. Here, we compare the interfacial properties of the N-terminal acetylated αS (Ac-αS) with non-acetylated αS (NH2-αS) at the air-water interface. Both the protein forms are highly surface-active, with surface pressure reaching up to ~30 mN/m upon compression. The pressure-area isotherms obtained from the repeated compression-expansion cycles display large hysteresis suggesting self-assembly at higher surface pressures. The expansion isotherm is characterized by a rapid decrease in surface pressure followed by a slower transition phase starting around 15-17 mN/m. These data suggest that the compressed monolayer breaks into small clusters upon expansion, followed by these clusters' loosening. The circular dichroism spectroscopic analysis of the Blodgett-deposited films suggests the protein to be in largely α-helical conformation. The linear dichroism investigations suggest the protein to be anisotropically deposited. Blodgett deposition of the Langmuir films, therefore, is a rather simple method for preparing oriented monolayers of surface-active macromolecules.

Polymyxin B accelerates the α-synuclein aggregation.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic neurons. It is characterised by the deposition of insoluble α-synuclein aggregates in the brain. Constipation is a common PD-associated condition, and the treatment of constipation with certain antibiotics seem to improve the PD symptoms. Polymyxin B, a last resort drug in treating the life-threatening Gram-negative bacterial infections, is one such antibiotic. The administration of polymyxin B in PD patients is known to alleviate the movement disorder symptoms; the mechanism of action, however, remains unclear. We, therefore, wondered if polymyxin B could modulate the aggregation of α-synuclein. We find that the polymyxin B catalyses the aggregation of α-synuclein into amyloid fibrils. At equimolar polymyxin B concentration, the lag phase was reduced to around one-third of that in the absence of polymyxin B.

Water-Alcohol Bigels from Fatty Acylated Dipeptides.

Peptide-based gels are emerging as an interesting class of biocompatible soft materials. 9-Fluorenylmethoxycarbonyl-protected amino acids and short peptides have gained considerable attention as promising gelators. Peptide amphiphiles, wherein an alkyl chain is appended to a polar peptidic moiety, are another important class of peptide-based gelators. Here, we report the alcohol/water bigels formed by the rather simple fatty acylated dipeptides wherein the peptidic moiety is made up of hydrophobic amino acids, viz., Val, Ile, and Leu. Lauroyl, myristoyl, and palmitoyl were investigated as the N-terminal fatty acyl groups. None of the lauroylated peptides caused gelation of methanol/water and ethanol/water mixtures up to 2 wt % peptide concentration. Eight out of the 27 peptides resulted in distinct bigels. The gels are composed of fibrous aggregates as characterized by electron microscopy. Infrared spectroscopy suggests the ß-sheet conformation of the peptidic region in the gels. Using the Ma-IV ethanol/water bigel as the representative gel, entrapment and steady release of the anticancer drug docetaxel are demonstrated. Such bigels from rather simple amphipathic peptides that are easily synthesized and purified through solvent extraction could be attractive gelator candidates with potential application in drug delivery.