Regulatory Role of ERG3 and Efg1 in Azoles-Resistant Strains of Candida albicans Isolated from Patients Diagnosed with Vulvovaginal Candidiasis Abstract Vulvovaginal candidiasis (VVC), caused by Candida albicans, affects women’s health and life. We aimed to explore the correlation between ERG3 as well as Efg1 mutation/overexpression and azoles-resistance, and the correlation between ERG3 and Efg1 mRNA expression in C. albicans. First, C. albicans was isolated from clinical VVC patients. ERG3 and Efg1 mutations were detected by polymerase chain reaction (PCR) and sequencing, and the expression levels of these two genes were also identified by qRT-PCR. Correlations between mutation/overexpression of ERG3/Efg1 and azoles-resistance as well as ERG3 and Efg1 mRNA expression were analyzed. Based on the ERG3 sequencing, the results showed that there were 2 missense mutation sites, 1 nonsense mutation site, and 4 silent mutation sites, while 1 missense mutation sites, 1 nonsense mutation site, and 12 silent mutation sites were found in Efg1. Furthermore, the mRNA levels of ERG3 gene in the strains sensitive to FCA, ITR or VRC were higher than those in the strains resistant to FCA, ITR, VRC (P < 0.05). While for the mRNA levels of Efg1, susceptible strains were lower than resistant strains. Besides, there was a significant linear negative correlation between ERG3 and Efg1 mRNA expression (r = − 0.614, P < 0.001).

Synthetic Biology Perspectives of Microbial Enzymes and Their Innovative Applications Abstract Microbial enzymes are high in demand and there is focus on their efficient, cost effective and eco-friendly production. The relevant microbial enzymes for respective industries needs to be identified but the conventional technologies don’t have much edge over it. So, there is more attention towards high throughput methods for production of efficient enzymes. The enzymes produced by microbes need to be modified to bear the extreme conditions of the industries in order to get prolific outcomes and here the synthetic biology tools may be augmented to modify such microbes and enzymes. These tools are applied to synthesize novel and efficient enzymes. Use of computational tools for enzyme modification has provided new avenues for faster and specific modification of enzymes in a shorter time period. This review focuses on few important enzymes and their modification through synthetic biology tools including genetic modification, nanotechnology, post translational modification.

Human Milk Microbiota: Transferring the Antibiotic Resistome to Infants Abstract Commensal bacterial population is believed to be a reservoir for antibiotic resistance genes (ARGs). The infant gut microbiota has relatively higher abundance of ARGs than the adults. These genes can get transferred from commensals to pathogens by horizontal gene transfer, which magnifies the spectrum of antibiotic resistance in the environment. The presence of ARGs in neo-nates and infants, with no prior antibiotic exposure, questions their origin in the naïve commensal population. Breast milk microbiota that is responsible for the initial seeding of infant gut microbiota has also been found to harbour a vast array of ARGs. This review discusses the recent findings that indicate the potential of breast milk microbiota to act as a vehicle for transmission of ARGs to infants.

Exploitation of Citrus Peel Extract as a Feedstock for Power Generation in Microbial Fuel Cell (MFC) Abstract Microbial fuel cells (MFCs) are envisioned as an evolving cost-effective process for treating organic wastes to simultaneously generate bioelectricity. Therefore, in present study a single chambered mediator- less air cathode MFC was operated for bioelectricity generation using citrus waste (CW) as a feedstock. The MFC was operated at four organic loading conditions (OLs; 3, 6, 9 and 12 kg/m3). The voltage generation and organic content reduction demonstrated the possibility of utilizing CW as a substrate in MFC. The polarization analysis revealed a high-power generation of 71.1 mW/m2 with low OL of 3 kg/m3. The decrease in pH and high volatile fatty acids (VFAs) generation was noted at high OL. Our current findings suggest better performance of MFC, in terms of energy generation and organic reduction at high OL.

Genome-Wide Analysis of Putative G-Quadruplex Sequences (PGQSs) in Onion Yellows Phytoplasma (Strain OY-M): An Emerging Plant Pathogenic Bacteria Abstract Phytoplasma, an emerging plant pathogen is an endocellular obligate parasite of plant phloem tissues with highly reduced genomes and low GC content. They contain a minimal set of genes essential for survival as an intracellular parasite. The role of G-Quadruplexes in pathogenicity has been reported in a variety of microbial pathogens. Detailed investigation on the genome wide occurrence and distribution of Putative G-Quadruplex forming Sequences (PGQSs) in the AT-rich genome of Onion yellows phytoplasma (strain OY-M) was carried out. Relative enrichment and depletion of these putative secondary structures in different genomic regions of OY-M was investigated with an aim to unravel their association with functionally important genomic locations. PGQSs density of 0.4407/Kbp was detected in the genome of OY-M phytoplasma, which is significantly higher than the average PGQSs density (0.136/Kbp) reported for other members of its phylum, namely Tenericutes. A non-random distribution of PGQSs across the length of the genome was observed. Putative promoter regions of OY-M were found to be particularly enriched in PGQSs followed by genic regions. The repeat rich regions were identified to have minimum PGQSs density. Presence of PGQSs in important genes such as those involved in secretory pathways of virulent factors, transport related functions, rRNA and tRNA was particularly intriguing. Our study reports for the first time a detailed investigation on the genome-wide locations of putative G-Quadruplexes in phytoplasma and highlights the need to further investigate their role in the metabolism and also in the mechanism of pathogenicity.

Isolation and Molecular Identification of Microsporidian Pathogen Causing Nosemosis in Muga Silkworm, Antheraea assamensis Helfer (Lepidoptera: Saturniidae ) Abstract Microsporidia are intracellular fungal parasites and they are the most common pathogens for sericulture. Microsporidian sp. can cause pebrine, a dreadful disease and lead to destructive disorder in Muga silkworm, Antheraea assamensis Helfer by vertical and horizontal transmission. This disease is the key factor obstructing the developmental progress of Muga culture in India. Nevertheless, molecular identification and characterization of pathogen associated with pebrine disease in A. assamensis is not yet established. Insect bioassay studies revealed that microsporidian infection in Muga silkworm, A. assamensis Helfer significantly reduced (P < 0.005) cocoon weight, pupal weight, shell weight and silk ratios. A new set of PCR primers suitable for amplification of small subunit ribosomal RNA (SSU-rRNA) of microsporidia infecting A. assamensis have been designed. The amplicon was cloned, sequenced and analysed. Microsporidia pathogen of wild silk moth A. assamensis has been identified at genus level as Nosema sp. AA1. Phylogeny of Nosema sp. AA1 was constructed on the basis of SSU-rRNA sequence and it has a close evolutionary relationship with microsporidian pathogens of other wild silkmoths. The arrangement and organization of the rRNA genes inferred that Nosema sp. AA1 belongs to true Nosema group and not to members of the Nosema/Vairimorpha group.

Solvent-Tolerant Acyltransferase from Bacillus sp. APB-6: Purification and Characterization Abstract Amidase from Bacillus sp. APB-6 with very good acyltransferase activity was purified to homogeneity with a purification fold of 3.68 and 53.20% enzyme yield. The purified protein's subunit molecular mass was determined approximately 42 kDa. Hyperactivity of the enzyme was observed at pH 7.5 (150 mM, potassium-phosphate buffer) and 50 °C of incubation. An enhancement in activity up to 42% was recorded with ethylenediaminetetraacetic acid and dithiothreitol. The kinetic parameter Km values for substrates: acetamide and hydroxylamine-hydrochloride were 73.0 and 153 mM, respectively. Further, the Vmax for acyltransferase activity was 1667 U/mg of protein and the Ki for acetamide was calculated as 37.0 mM. The enzyme showed tolerance to various organic solvents (10%, v/v) and worked well in the biphasic reaction medium. The acyltransferase activity in presence of solvents i.e. biphasic medium may prove highly favorable for the transformation of hydrophobic amides, which otherwise is not possible in simple aqueous phase.

Antiherpetic Effect of Topical Formulations Containing Sulfated Polysaccharide from Adenanthera pavonina Abstract Adenanthera pavonina is a native tree of Africa and Asia, introduced in Brazil for reforestation and wood industry. Several pharmacological activities have described scientifically, including antiviral activity. This study evaluated the antiviral effect of sulfated polysaccharide of Adenanthera pavonina (SPAp) against acyclovir (ACV)—resistant (AR-29) and sensitive (KOS) herpes simplex virus strains. The 50% cytotoxic concentration (CC50) was determined by MTT method and the 50% inhibitory concentration (IC50) was evaluated by plaque reduction assay. The in vivo SPAp antiviral activity was performed in Balb/c mice infected by skin scarification and treated with topical 0.5% (w/w) SPAp formulations. SPAp showed a CC50 of 47.81 μg/mL and the IC50 were 0.49 μg/mL (SI = 97.5) and 0.54 μg/mL (SI = 88.5) for the strains KOS and AR-29, respectively. Our results demonstrated that mice treated with SPAp presented a delay in the development and progression of skin lesions compared with the control group.

Construction of a Lactobacillus plantarum Strain Expressing the Capsid Protein of Porcine Circovirus Type 2d (PCV2d) as an Oral Vaccine Abstract Porcine circovirus type 2 (PCV2) is a pathogenic virus that causes high rates of porcine death, resulting in severe economic losses to the swine industry. In recent years, the prevalence of PCV2d genotype infection in pigs has increased, but most commercially available vaccines were developed against the PCV2a strain and do not ensure complete protection from PCV2d. Here, we first constructed an expression vector for the antigenic ORF2-encoded capsid protein of PCV2d (pLp3050-His6-tag-capsid). We then utilized Lactobacillus plantarum to express the protein at mucosal sites in orally vaccinated mice. After transducing L. plantarum with pLp3050-His6-tag-capsid, the expressed protein could be found in cell wall and cell-free supernatant fractions by Western blotting. Using flow cytometry, we found that L. plantarum cells with surface-displayed capsid protein increased with time after SppIP induction. Finally, mice that were orally immunized 18 times with capsid-expressing L. plantarum showed increased levels of capsid-specific sIgA and virus neutralizing activity at mucosal sites, suggesting mucosal immunity had been stimulated by the vaccine. Overall, our findings demonstrate the feasibility and utility of a PCV2d-based vaccine, which may be of great value in porcine agriculture.

Hetero-Polysaccharides Secreted from Dunaliella salina Exhibit Immunomodulatory Activity Against Peripheral Blood Mononuclear Cells and RAW 264.7 Macrophages Abstract Several species of microalgae have been known to produce exopolysaccharides (EPS) with potential immune activity. In the present investigation, ethyl acetate fraction of crude EPS secreted by Dunaliella salina was explored for immunomodulatory activity against peripheral blood mononuclear cells (PBMC) and RAW 264.7 macrophages. Effect of EPS on cell growth and cytokines production were measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and ELISA respectively. Griess reagent was used for measuring the nitric oxide production in RAW 264.7 macrophages. FTIR analysis and mass spectroscopy were carried out for the characterization. Ethyl acetate fraction exhibited dose dependent increase in proliferative index and cytokines production (IFN-γ, TNF-α, TGF-β). At low concentration (250 and 500 µg/mL), it showed growth inhibition and at higher concentration (1000 and 1500 µg/mL), it enhanced the cell growth. Interestingly, the pronounced increased TNF-α production was observed in ethyl acetate fraction treated PBMC cells at higher concentration (750 and 1000 µg/mL) indicating the immunostimulatory effect. In RAW cells, concentration dependent diminished cell growth (IC50 = 691 µg/mL) and nitric oxide production (IC50 = 630 µg/mL) was observed. FTIR analysis showed the presence of polysaccharides due to the detection of hydroxyl (–OH), Carbonyl (C–O) and alkyl (C–H) groups. Mass spectroscopy results revealed ethyl acetate fraction as penta-saccharide (m/z = 887.56 and 886.54) which are confirmed to be hetero-polysaccharides consisting of hexoses and pentoses along with association of ions. These results suggest that penta-saccharide (ethyl acetate fraction) isolated from D. salina may have the potential to be used for therapeutic purpose as immunomodulatory agent.