Capping Protein Research Articles

Fast and efficient template-mediated synthesis of genetic variants.

Efficient methods for the generation of specific mutations enable the study of functional variations in natural populations and lead to advances in genetic engineering applications. Here, we present a new approach, mutagenesis by template-guided amplicon assembly (MEGAA), for the rapid construction of kilobase-sized DNA variants. With this method, many mutations can be generated at a time to a DNA template at more than 90% efficiency per target in a predictable manner. We devised a robust and iterative protocol for an open-source laboratory automation robot that enables desktop production and long-read sequencing validation of variants. Using this system, we demonstrated the construction of 31 natural SARS-CoV2 spike gene variants and 10 recoded Escherichia coli genome fragments, with each 4 kb region containing up to 150 mutations. Furthermore, 125 defined combinatorial adeno-associated virus-2 cap gene variants were easily built using the system, which exhibited viral packaging enhancements of up to 10-fold compared with wild type. Thus, the MEGAA platform enables generation of multi-site sequence variants quickly, cheaply, and in a scalable manner for diverse applications in biotechnology.

Integrated molecular and quantum mechanical approach to identify novel potent natural bioactive compound against 2′-O-methyltransferase (nsp16) of SARS-CoV-2

With the advent of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak, efforts are still in progress to find out a functional cure for the infection. Among the various protein targets, nsp16 capping protein is one of the vital targets for drug development as it protects the virus against the host cell nucleases and evading innate immunity. The nsp16 protein forms a heterodimer with a co-factor nsp10 and triggers 2′-O-methyltransferase activity which catalyzes the conversion of S-adenosyl methionine into S-adenosyl homocysteine. The free methyl group is transferred to the 2′-O position on ribose sugar at the 5′ end of mRNA to form the cap-1 structure which is essential for replication of the virus and evading the innate immunity of the host. In this study, we identify a potential lead natural bioactive compound against nsp16 protein by systematic cheminformatic analysis of more than 144k natural compounds. Virtual screening, molecular docking interactions, ADMET profiling, molecular dynamics (MD) simulations, molecular mechanics-generalized born surface area (MM-GBSA), free energy analysis and density functional theory analysis were used to discover the potential lead compound. Our investigation revealed that ZINC8952607 (methyl-[(6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-yl)aminomethyl]BLAHone) has the greatest binding affinity and best pharmacokinetic parameters due to presence of carbazol and BLAHone (biaryl moiety). Further, time-dependent MD simulation analysis substantiates the stability and rigidness of nsp16 protein even after interaction with the lead compound. We believe that the compound ZINC8952607 might establish as a novel natural drug candidate against CoVID-19 infection. Communicated by Ramaswamy H. Sarma

Characterization of novel cell-penetrating peptides derived from the capsid protein of beak and feather disease virus

Beak and feather disease virus (BFDV) is a 17-20 nm icosahedral virus belonging to the Circoviridae family. Psittacine beak and feather disease (PBFD) is caused by BFDV and its common symptoms include abnormal feather, beak, and claw development, as well as immunosuppression in various bird species. In this study, novel cell-penetrating peptides (CPPs) in the capsid protein (Cap) of BFDV were identified through bioinformatic analyses, after which they were experimentally characterized. The cell-penetrating activities of both CPP1 and CPP2 of BFDV were analyzed through flow cytometry and image analysis. The internalization of CPP1 and CPP2 was both dose- and time-dependent but their uptake efficiencies varied depending on the cell type. The cell-penetrating activities of BFDV CPP1 and CPP2 were both superior to that of a typical CPP-TAT originating from the viral protein of human immunodeficiency virus. The cellular uptake of 5 μM CPP1 was close to that of 25 μM TAT, albeit with less cytotoxicity. Using the identified CPPs, the pc-mCheery, pc-Rep, and pc-Cap plasmids were successfully delivered into the target cells for expression. Moreover, both the replication-associated protein with the tag and the Cap protein with the tag could also be successfully delivered into the cells by CPP1 and CPP2. Multiple endocytosis pathways and direct translocation were involved in the cell internalization of CPP1 and CPP2. Furthermore, the delivery of the apoptin gene using CPP1 and CPP2 effectively triggered apoptosis, thus confirming the potential of these CPPs as delivery vehicles. Similarly, green fluorescent protein (GFP) fused with CPP1 or CPP2 at their N-terminus successfully entered the cells. However, the cell internalization efficiency of CPP2-GFP was higher than that of CPP1-GFP. Taken together, our findings demonstrated that both CPP1 and CPP2 of BFDV have promising potential as novel CPPs.

Development of a recombinant reporter Getah virus for antiviral drug screening assays

Getah virus (GETV), is an often neglected and re-emerging mosquito-borne RNA virus. GETV can cause illness accompanied with high fever, rash, incapacitating arthralgia and chronic arthritis or encephalitic disease in affected animals. Currently, there is no specific treatment or vaccine against GETV infection. In this study, we developed three recombinant viruses by inserting different reporter protein genes between the Cap and pE2 genes. The reporter viruses exhibited high replication capacity similar to the parental virus. The rGECiLOV and rGECGFP viruses were genetically stable within at least ten rounds of passages in BHK-21 cells. We confirmed that the reporter virus, rGECGFP, facilitated the antiviral assays against GETV by testing it with the known inhibitor, ribavirin. It was also found that the compound, doxycycline, showed an inhibitory effect on GETV replication. In addition, rGECGFP was found to be an authentic mimic of the parental virus infection in 3-day-old mice, but with milder pathogenicity. The reporter viruses will contribute to the assessment of viral replication and proliferation, tracking and elucidating of alphavirus-host interactions. In addition, they will help in the screening of potential antiviral compounds.

Phylogenetic and Structural Analysis of Porcine Circovirus Type 2 from 2016 to 2021 in Jilin Province, China.

Porcine circovirus disease (PCVD) caused by porcine circovirus type 2 (PCV2) is widely distributed in pig farms. Up until now, nine genotypes of PCV2, PCV2a to 2i, have been identified in diseased pigs worldwide. This study analyzed 302 samples collected in the Jilin Province of China from 2016 to 2021, followed by genetic analysis of the PCV2 isolates. Meanwhile, the antigen epitopes, amino acid mutations, 3D structure of the PCV2 isolates and commercially available vaccine strains were evaluated and compared. The results showed that the predominant genotypes of PCV2 were PCV2b, followed by PCV2e and PCV2d in Jilin Province during 2016-2021. Although mutations were detected in the isolates, no recombination occurred in the PCV2 isolates, indicating a stable genotype of PCV2 in Jilin Province during these years. Moreover, the B cell epitopes in the Cap and Rep proteins of eighteen PCV2 isolates and T cell epitopes in the Cap of the isolates were changed compared to three currently used vaccine strains. The mutations in the Cap and Rep proteins did not affect their spatial conformation. Therefore, bivalent or multivalent vaccines with different genotypes of PCV2 might improve the protective effect of vaccines.

203 Novel Adeno-Associated Viral (AAV) Vectors for Preferential Targeting of Peripheral Nerve Tumors

INTRODUCTION: Definitive treatment for peripheral nerve tumors by surgical excision is associated with significant morbidity. Gene therapy with adeno-associated viral (AAV) vectors is emerging as a promising therapy for these tumors. Directed evolution is a technique that involves applying selective pressure to large libraries of mutated cap genes to bioengineer AAV vectors that selectively bind to and transduce schwannoma tissue. METHODS: Tumor samples were harvested from 8 patients and divided for cell culture. Cultures were infected with novel AAV vector C1, novel AAV vector C5, AAV1, AAV2, and AAV6 at MOIs of 2500, 5000, and 10000 for 3-, 7-, and 14-day periods. GFP fluorescence was used to quantify transduction efficiency of each vector. Statistical analysis was performed with one-way ANOVA and student t test. RESULTS: AAV2 had significantly higher transduction efficiency at 65% +/- 11 compared to all other vectors (p < 0.001). The remaining order of highest transduction efficiency was novel AAV vector C2 at 21% +/- 4, novel AAV vector C5 at 12% +/- 3, AAV1 at 7% +/- 2, and AAV6 at 6% +/- 2. Higher transduction efficiencies were seen at higher MOIs (2500 vs. 5000 vs. 10000) with no significant difference in cell count (2500: 74.25 +/- 25.59. 5000: 105.50 +/- 15.93, 10000: 90.75 +/- 5.74, p = 0.094). Longer post-infection incubation periods (3-day vs. 7-day vs. 14-day) led to higher transduction efficiencies. CONCLUSIONS: AAV2 remains the gold standard for delivery of gene therapy to peripheral nerve tissue. Higher MOIs and longer incubation periods can better differentiate transduction efficiency when comparing multiple AAV vectors without having detrimental effects on cell culture.

Molecular detection and genetic characterization of porcine circovirus 4 (PCV4) in Thailand during 2019–2020

Porcine circovirus 4 (PCV4) is considered a novel PCV, firstly found in China in 2019 and later discovered in Korea. This present study investigated the prevalence and genetic characteristics of PCV4 from high pig-density areas in Thailand during 2019–2020. From 734 samples, three samples (0.4%) from aborted fetuses and porcine respiratory disease complex (PRDC) cases were found positive for PCV4, two of the PCV4-positive samples were coinfected with both PCV2 and PRRSV, and the other PCV4-positive sample was found coinfected with PCV2. In situ hybridization (ISH) revealed the presence of PCV4 in the bronchial epithelial cells and in lymphocytes and histiocyte-like cells in the lymphoid follicles of the PRDC-affected pig. The complete Thai PCV4 genome had over 98% nucleotide identity with other PCV4 strains and was closely related to the Korean and Chinese PCV4b strains. Importantly, the amino acid residue at position 212 of the Cap gene is recommended for differentiating PCV4a (212L) from PCV4b (212M) based on currently available PCV4 genome sequences. These findings provide important clues for the pathogenesis, epidemiology, and genetic characteristics of PCV4 in Thailand.

The function of plant PR1 and other members of the CAP protein superfamily in plant-pathogen interactions.

The pathogenesis-related (PR) proteins of plants have originally been identified as proteins that are strongly induced upon biotic and abiotic stress. These proteins fall into 17 distinct classes (PR1-PR17). The mode of action of most of these PR proteins has been well characterized, except for PR1, which belongs to a widespread superfamily of proteins that share a common CAP domain. Proteins of this family are not only expressed in plants but also in humans and in many different pathogens, including phytopathogenic nematodes and fungi. These proteins are associated with a diverse range of physiological functions. However, their precise mode of action has remained elusive. The importance of these proteins in immune defence is illustrated by the fact that PR1 overexpression in plants results in increased resistance against pathogens. However, PR1-like CAP proteins are also produced by pathogens and deletion of these genes results in reduced virulence, suggesting that CAP proteins can exert both defensive and offensive functions. Recent progress has revealed that plant PR1 is proteolytically cleaved to release a C-terminal CAPE1 peptide, which is sufficient to activate an immune response. The release of this signalling peptide is blocked by pathogenic effectors to evade immune defence. Moreover, plant PR1 forms complexes with other PR family members, including PR5, also known as thaumatin, and PR14, a lipid transfer protein, to enhance the host's immune response. Here, we discuss possible functions of PR1 proteins and their interactors, particularly in light of the fact that these proteins can bind lipids, which have important immune signalling functions.

Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation

Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed. Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number. Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells. Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation.

In vivo characterization of the critical interaction between the RNA exosome and the essential RNA helicase Mtr4 in Saccharomyces cerevisiae.

The RNA exosome is a conserved molecular machine that processes/degrades numerous coding and non-coding RNAs. The 10-subunit complex is composed of three S1/KH cap subunits (human EXOSC2/3/1; yeast Rrp4/40/Csl4), a lower ring of six PH-like subunits (human EXOSC4/7/8/9/5/6; yeast Rrp41/42/43/45/46/Mtr3), and a singular 3'-5' exo/endonuclease DIS3/Rrp44. Recently, several disease-linked missense mutations have been identified in structural cap and core RNA exosome genes. In this study, we characterize a rare multiple myeloma patient missense mutation that was identified in the cap subunit gene EXOSC2. This missense mutation results in a single amino acid substitution, p.Met40Thr, in a highly conserved domain of EXOSC2. Structural studies suggest that this Met40 residue makes direct contact with the essential RNA helicase, MTR4, and may help stabilize the critical interaction between the RNA exosome complex and this cofactor. To assess this interaction in vivo, we utilized the Saccharomyces cerevisiae system and modeled the EXOSC2 patient mutation into the orthologous yeast gene RRP4, generating the variant rrp4-M68T. The rrp4-M68T cells show accumulation of certain RNA exosome target RNAs and show sensitivity to drugs that impact RNA processing. We also identified robust negative genetic interactions between rrp4-M68T and specific mtr4 mutants. A complementary biochemical approach revealed that Rrp4 M68T shows decreased interaction with Mtr4, consistent with these genetic results. This study suggests that the EXOSC2 mutation identified in a multiple myeloma patient impacts the function of the RNA exosome and provides functional insight into a critical interface between the RNA exosome and Mtr4.

Cytoskeletal association of ATP citrate lyase controls the mechanodynamics of macropinocytosis

Macropinocytosis is an actin-dependent mode of nonselective endocytosis that mediates the uptake of extracellular fluid-phase cargoes. It is now well recognized that tumor cells exploit macropinocytosis to internalize macromolecules that can be catabolized and used to support cell growth and proliferation under nutrient-limiting conditions. Therefore, the identification of molecular mechanisms that control macropinocytosis is fundamental to the understanding of the metabolic adaptive landscape of tumor cells. Here, we report that the acetyl-CoA-producing enzyme, ATP citrate lyase (ACLY), is a key regulator of macropinocytosis and describes a heretofore-unappreciated association of ACLY with the actin cytoskeleton. The cytoskeletal tethering of ACLY is required for the spatially defined acetylation of heterodimeric actin capping protein, which we identify as an essential mediator of the actin remodeling events that drive membrane ruffling and macropinocytosis. Furthermore, we identify a requirement for mitochondrial-derived citrate, an ACLY substrate, for macropinocytosis, and show that mitochondria traffic to cell periphery regions juxtaposed to plasma membrane ruffles. Collectively, these findings establish a mode of metabolite compartmentalization that supports the spatiotemporal modulation of membrane-cytoskeletal interactions required for macropinocytosis by coupling regional acetyl-CoA availability with dynamic protein acetylation.

Deciphering the roles of cell shape and Fat and Dachsous planar polarity in arranging the Drosophila apical microtubule network through quantitative image analysis.

In epithelial cells, planar polarization of subapical microtubule networks is thought to be important for both breaking cellular symmetry and maintaining the resulting cellular polarity. Studies in the Drosophila pupal wing and other tissues have suggested two alternative mechanisms for specifying network polarity. On one hand, mechanical strain and/or cell shape have been implicated as key determinants; on the other hand, the Fat-Dachsous planar polarity pathway has been suggested to be the primary polarizing cue. Using quantitative image analysis in the pupal wing, we reassess these models. We found that cell shape was a strong predictor of microtubule organization in the developing wing epithelium. Conversely, Fat-Dachsous polarity cues do not play any direct role in the organization of the subapical microtubule network, despite being able to weakly recruit the microtubule minus-end capping protein Patronin to cell boundaries. We conclude that any effect of Fat-Dachsous on microtubule polarity is likely to be indirect, via their known ability to regulate cell shape.

First Molecular Detection and Genetic Analysis of a Novel Porcine Circovirus (Porcine Circovirus 4) in Dogs in the World.

A novel circovirus species was identified in farmed pigs and designated porcine circovirus 4 (PCV4); it has recently been proved to be pathogenic to piglets. However, little is known about its cross-species transmission, and there is no evidence of PCV4 in dogs. A total of 217 fecal samples were collected from diarrheal dogs in Henan Province, China, and tested for the presence of PCV4 using a real-time PCR assay. Among the 217 samples, the total positivity rate for PCV4 was 5.99% (13/217 samples), with rates of 7.44% and 4.17% in 2020 and 2021, respectively. PCV4 was detected in dogs in 6 of 10 cities, demonstrating that PCV4 could be detected in dogs in Henan Province, China. One PCV4 strain (HN-Dog) was sequenced in this study and shared high levels of identity (97.9% to 99.6%) with reference strains at the genome level. Phylogenetic analysis based on complete genome sequences of HN-Dog and 42 reference strains showed that the HN-Dog strain was closely related to 3 PCV4 reference strains (from pig, raccoon dog, and fox) but differed genetically from other viruses in the genus Circovirus. Three genotypes, i.e., PCV4a, PCV4b, and PCV4c, were confirmed by phylogenetic analysis of complete genome sequences of 42 PCV4 strains, and one amino acid variation in Rep protein (V239L) and three amino acid variations in Cap protein (N27S, R28G, and M212L) were considered conserved genotype-specific molecular markers. In conclusion, the present study is the first to report the discovery of the PCV4 genome in dogs, and the association between PCV4 infection and diarrhea warrants further study. IMPORTANCE This study is the first to report the presence of PCV4 in dogs worldwide, and the first complete genome sequence was obtained from a dog affected with diarrhea. Three genotypes of PCV4 strains (PCV4a, PCV4b, and PCV4c) were determined, as supported by specific amino acid markers (V239L for open reading frame 1 [ORF1] and N27S R28G and M212L for ORF2). These findings help us understand the current status of intestinal infections in pet dogs in Henan Province, China, and also prompted us to accelerate research on the pathogenesis, epidemiology, and cross-species transmission of PCV4.

Evolutionary tuning of barbed end competition allows simultaneous construction of architecturally distinct actin structures.

How cells simultaneously assemble actin structures of distinct sizes, shapes, and filamentous architectures is still not well understood. Here, we used budding yeast as a model to investigate how competition for the barbed ends of actin filaments might influence this process. We found that while vertebrate capping protein (CapZ) and formins can simultaneously associate with barbed ends and catalyze each other's displacement, yeast capping protein (Cap1/2) poorly displaces both yeast and vertebrate formins. Consistent with these biochemical differences, in vivo formin-mediated actin cable assembly was strongly attenuated by the overexpression of CapZ but not Cap1/2. Multiwavelength live cell imaging further revealed that actin patches in cap2∆ cells acquire cable-like features over time, including recruitment of formins and tropomyosin. Together, our results suggest that the activities of S. cerevisiae Cap1/2 have been tuned across evolution to allow robust cable assembly by formins in the presence of high cytosolic levels of Cap1/2, which conversely limit patch growth and shield patches from formins.

Myosin-I facilitates symmetry breaking and promotes the growth of actin 'comet tails'.

Myosin-Is are single-headed, membrane associated members of the myosin superfamily that participate in crucial cellular processes related to membrane morphology and trafficking. Recent studies show that myosin-I isoforms frequently concentrate on membranes in areas of Arp2/3 complex-mediated actin polymerization that affect membrane shape and dynamics. To investigate how myosin-Is affect actin assembly, we performed a “comet tail assay” where branched actin networks were nucleated by Arp2/3 complex from a bead surface coated with a nucleation promoting factor (NPF). Actin filaments first formed a cloud around the bead, which transitioned into a polarized comet tail after symmetry breaking. We site-specifically coupled a range of densities of myosin-Is to the bead surface and assessed their effects on actin polymerization, network architecture, and symmetry breaking. We found that high myosin densities prevented comet tail formation. Instead, an extremely sparse actin cloud was created surrounding the bead. Decreasing the myosin density resulted in an actin network that broke symmetry more rapidly and formed a polarized comet tail. This actin comet tail was able to elongate from the bead at a faster rate and frequently showed smooth, rather than pulsatile motion. Myosin also changed the architecture of actin networks in comet tails. Compared with the coherent actin networks from non-myosin-coated beads, actin networks emerging from myosin-coated beads were sparser and more disordered, which might be due to the gliding and rotating effect of myosin. Strikingly, under low capping protein concentrations, where bead was embedded in a highly-dense actin shell and symmetry breaking was completely inhibited, myosin-coated beads were able to overcome this inhibition to break symmetry and form a comet tail. These studies show synergy between myosin activity and actin polymerization to power morphological changes at the cell membrane.

Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022

African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are important DNA viruses that cause reproductive disorders in sows, which result in huge losses in pig husbandry, especially in China. The multiplex qPCR assay could be utilized as a simultaneous diagnostic tool for field-based surveillance and the control of ASFV, PCV2, and PRV. Based on the conserved regions on the p72 gene of ASFV, the Cap gene of PCV2, the gE gene of PRV, and the porcine endogenous β-Actin gene, the appropriate primers and probes for a multiplex TaqMan real-time PCR test effective at concurrently detecting three DNA viruses were developed. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to swine reproductive diseases. In addition, its sensitivity was great, with a detection limit of 101 copies/L of each pathogen, and its repeatability was excellent, with intra- and inter-group variability coefficients of <2%. Applying this assay to detect 383 field specimens collected from 2020 to 2022, the survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV + PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. Overall, the assay established in this study provides an effective tool for quickly distinguishing the viruses causing sow reproductive disorders, suggesting its huge clinical application value in the diagnosis of swine diseases.

Thermomyces lanuginosus: A prospective thermophilic fungus for green synthesis and stabilization of BioAgNPs through glucoamylase

HypothesisThe rise of bionanoscience has led to the use of silver nanoparticles in therapeutic and industrial applications like biomedicine, biocatalysis, bioremediation and biosensing. Enhancement of metal nanoparticle stability is of critical significance when it comes to safe handling and efficient use of nanoparticles for various applications. In this regard, thermophilic fungi is a potential candidate with their massive thermostable excretome titres. This work aims to explore the potential of thermophilic fungi to provide surface stability to biogenic silver nanoparticles (BioAgNPs). ExperimentThermophilic fungus Thermomyces lanuginosus STm was used to synthesize BioAgNPs. The size and shape of BioAgNPs were characterized using ultraviolet–visible spectroscopy, transmission electron microscopy (TEM) and X-ray diffraction techniques. The most significant process variables affecting mycosynthesis of BioAgNPs were screened using statistical experimental design of Placket Berman model. The mechanism of mycosynthesis and capping on biogenic nanoparticles was explored using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography–mass spectrometry (LC-MS/MS). FindingsCharacterization of BioAgNPs by ultraviolet–visible spectroscopy revealed surface plasmon resonance at 410 nm, X-ray diffraction calculated 7–24 nm diameter of BioAgNPs and transmission electron microscopy showed BioAgNPs to be spherical with 5–35 nm size range. The physico-chemical factors affecting synthesis of BioAgNPs were screened by statistical modeling using Placket Berman experiment design which revealed the significant role of pH and silver precursor salt concentration on BioAgNPs synthesis. The supramolecular bound protein moiety was initially screened using FTIR analysis. The protein coating around BioAgNPs was found to be 67 kDa glucoamylase when detached from NP and investigated using SDS-PAGE and LCMS/MS analysis. In vitro cytotoxic assay against eukaryotic animal model Artemia salina (brine shrimp) larvae revealed dose dependent decrease in viability indicating BioAgNPs to be less cytotoxic, as compared to silver in ionic form. ConclusionGlucoamylase protein (67 kDa) secreted extracellularly by T. lanuginosus STm was discovered to be the capping protein surrounding BioAgNPs. It is interesting to note that in case of BioAgNPs synthesized by extracellular titre of thermophilic fungus, the adsorbed thermostable protein glucoamylase can possibly help in retaining activity of BioAgNPs at elevated temperature as per the industrial requirement. Thus it can be a natural way of immobilization of enzyme on the surface of nanoparticles while eliminating the extra steps require for immobilizing for commercial uses. In terms of acute toxicity, BioAgNPs were found to be less toxic and safer as compared to ionic silver.

Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal.

Adeno-associated viruses (AAV) are important vectors for gene therapy, and accordingly, many aspects of their cell transduction pathway have been well characterized. However, the specific mechanisms that AAV virions use to enter the host nucleus remain largely unresolved. We therefore aimed to reveal the interactions between the AAV Cap protein and the nuclear transport protein importin alpha (IMPα) at an atomic resolution. Herein we expanded upon our earlier research into the Cap nuclear localization signal (NLS) of a porcine AAV isolate, by examining the influence of upstream basic regions (BRs) towards IMPα binding. Using a high-resolution crystal structure, we identified that the IMPα binding determinants of the porcine AAV Cap comprise a bipartite NLS with an N-terminal BR binding at the minor site of IMPα, and the previously identified NLS motif binding at the major site. Quantitative assays showed a vast difference in binding affinity between the previously determined monopartite NLS, and bipartite NLS described in this study. Our results provide a detailed molecular view of the interaction between AAV capsids and the nuclear import receptor, and support the findings that AAV capsids enter the nucleus by binding the nuclear import adapter IMPα using the classical nuclear localization pathway.

The Effect of Sub-Lethal Successive Applications of Photodynamic Therapy on Candida albicans Biofilm Depends on the Photosensitizer.

This study aimed to evaluate the potential of successive applications of sub-lethal doses of the antimicrobial photodynamic therapy (aPDT) mediated by Photodithazine® (PDZ) and curcumin (CUR) associated with LED in the viability, reactive oxygen species (ROS) production, and gene expression of Candida albicans. The microbial assays were performed using planktonic cultures and biofilms. Ten successive applications (Apl#) were performed: aPDT (P+L+; C+L+), photosensitizer (P+L-; C+L-), and LED (P-L+; C-L+). Control groups were used (P-L-; C-L-). The viability of C. albicans was determined by cultivating treated cultures on agar plates with or without fluconazole (FLU). In addition, the ROS detection and expression of SOD1, CAP1, and ERG11 genes were determined. For planktonic cultures, no viable colonies were observed after Apl#3 (without FLU) and Apl#2 (with FLU) for either photosensitizer. Biofilm treated with P+L+ resulted in the absence of cell viability after Apl#7, while C+L+ showed ~1.40 log10 increase in cell viability after Apl#2, regardless of FLU. For both photosensitizers, after the last application with viable colonies, the production of ROS was higher in the biofilms than in the planktonic cultures, and SOD1 expression was the highest in P+L+. A reduction of CAP1 and ERG11 expression occurred after P+L+, regardless of FLU. C+L+ had a higher level of ROS, and the treatments were non-significant for gene expression. Sub-lethal doses of aPDT mediated by CUR could induce C. albicans resistance in biofilms, while C. albicans cells in biofilms were susceptible to aPDT mediated by PDZ.

Application of silver nanoparticles synthesized through varying biogenic and chemical methods for wastewater treatment and health aspects.

Nanotechnology uses biological and non-biological materials to create new systems at the nanoscale level. In recent years, the use of silver nanomaterials has attracted worldwide attention thanks to their wide range of applications as catalysts in several environmental processes including the degradation of organic pollutants and medicinal biotechnology. This study reports the synthesis of silver nanoparticles (AgNPs) through different methods including the biogenic methods based on leaf extract of Conocarpus erectus and a bacterial strain Pseudomonas sp. as well as chemically based abiotic method and comparison of their dye degradation potential. The synthesis of AgNPs in all samples was confirmed by UV-visible spectroscopy peaks at 418-420nm. Using scanning electrom microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray differaction (XRD), and X-ray photoelectron spectroscopy (XPS), the biologically synthesized AgNPs were characterized as spherical shape of material with capping proteins that were involved in the stabilization of nanoparticles (NPs). The biologically synthesized AgNPs showed higher degradation (< 90%) of dyes as compared to chemically synthesized NPs. A prominent reduction of total dissolved solids (TDS), electrical conductivity (EC), pH, and chemical oxygen demand (COD) in textile wastewater spiked with reactive black 5 and reactive red 120 was observed by biologically synthesized AgNPs. AgNPs synthesized by Conocarpus erectus and Pseudomonas sp. also showed better characteristic anticancer and antidiabetic activities as compared to chemically synthesized ones. The results of this study suggested that C. erectus and Pseudomonas sp. based AgNPs can be exploited as an eco-friendly and cost-efficient materials to treat the wastewater and potential other polluted environments as well as to serve the medicinal field.