Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pseudomonas fluorescens in Raw Milk

Raw milk is susceptible to microbial contamination during transportation and storage. Pseudomonas fluorescens producing heat-resistant enzymes have become the most common and harmful psychrophilic microorganisms in the cold chain logistics of raw milk. To rapidly detect P. fluorescens in raw milk, the protease gene aprX was selected as a detection target to construct a set of primers with strong specificity, and a loop-mediated isothermal amplification (LAMP) assay was established. The detection thresholds of the LAMP assay for pure cultured P. fluorescens and pasteurized milk were 2.57 × 102 and 3 × 102 CFU/mL, respectively. It had the advantages over conventional method of low detection threshold, strong specificity, rapid detection, and simple operation. This LAMP assay can be used for online monitoring and on-site detection of P. fluorescens in raw milk to guarantee the quality and safety of dairy products.

Quality Control of Mitochondria in Parkinson's Disease (PD) Using ATP And Bradford Assays

Parkinson's disease is a heterogeneous, multifactorial and often complex disease characterized by motor impairment due to the presence of Lewy bodies and prominent degeneration of dopaminergic neurons in the substantia nigra. Although the specific pathogenesis involving PD remains under investigation, mitochondrial dysfunction has been widely accepted as one of the major pathogenic pathways underlying the development of PD. Based on the hypothesis that depiction of HtrA2 (serine protease gene, mitochondrial precursor) might contribute to an increase in mitochondrial stress and transcriptional upregulation of the nuclear stress-response CHOP gene. The present study aimed to analyze through laboratory-based research the role of HtrA2 and CHOP in the transmission of stress signaling and the consequent activation of mitochondrial quality control in Parkinson's disease using ATP and Bradford assays.

Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis

In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulfhydryl groups exerted significant effects, strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibited the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the Merops peptidase database.

Co-option of an extracellular protease for transcriptional control of nutrient degradation in the fungus Aspergillus nidulans

Nutrient acquisition is essential for all organisms. Fungi regulate their metabolism according to environmental nutrient availability through elaborate transcription regulatory programs. In filamentous fungi, a highly conserved GATA transcription factor AreA and its co-repressor NmrA govern expression of genes involved in extracellular breakdown, uptake, and metabolism of nitrogen nutrients. Here, we show that the Aspergillus nidulans PnmB protease is a moonlighting protein with extracellular and intracellular functions for nitrogen acquisition and metabolism. PnmB serves not only as a secreted protease to degrade extracellular nutrients, but also as an intracellular protease to control the turnover of the co-repressor NmrA, accelerating AreA transcriptional activation upon nitrogen starvation. PnmB expression is controlled by AreA, which activates a positive feedback regulatory loop. Hence, we uncover a regulatory mechanism in the well-established controls determining the response to nitrogen starvation, revealing functional evolution of a protease gene for transcriptional regulation and extracellular nutrient breakdown.

Characterization of a Novel Aspartic Protease from Rhizomucor miehei Expressed in Aspergillus niger and Its Application in Production of ACE-Inhibitory Peptides

Rhizomucor miehei is an important fungus that produces aspartic proteases suitable for cheese processing. In this study, a novel aspartic protease gene (RmproB) was cloned from R. miehei CAU432 and expressed in Aspergillus niger. The amino acid sequence of RmproB shared the highest identity of 58.2% with the saccharopepsin PEP4 from Saccharomyces cerevisiae. High protease activity of 1242.2 U/mL was obtained through high density fermentation in 5 L fermentor. RmproB showed the optimal activity at pH 2.5 and 40 °C, respectively. It was stable within pH 1.5–6.5 and up to 45 °C. RmproB exhibited broad substrate specificity and had Km values of 3.16, 5.88, 5.43, and 1.56 mg/mL for casein, hemoglobin, myoglobin, and bovine serum albumin, respectively. RmproB also showed remarkable milk-clotting activity of 3894.1 SU/mg and identified the cleavage of Lys21-Ile22, Leu32-Ser33, Lys63-Pro64, Leu79-Ser80, Phe105-Met106, and Asp148-Ser149 bonds in κ-casein. Moreover, duck hemoglobin was hydrolyzed by RmproB to prepare angiotensin-I-converting enzyme (ACE) inhibitory peptides with high ACE-inhibitory activity (IC50 of 0.195 mg/mL). The duck hemoglobin peptides were further produced at kilo-scale with a yield of 62.5%. High-level expression and favorable biochemical characterization of RmproB make it a promising candidate for cheese processing and production of ACE-inhibitory peptides.

Occurrence of Sclerotinia sclerotiorum causing Sclerotinia root rot on American ginseng in northeastern China

American ginseng (Panax quinquefolium) is a valuable medicinal plant that is commercially cultivated in China. In May 2020, Sclerotinia root rot of American ginseng was observed on 4-year-old plants in Fusong County in northeastern China, which is the most important part of the country for American ginseng cultivation. The pathogen only infected the tuberous ginseng roots, with sclerotia tightly attached to the root surface. Infected roots, which were brownish and had a watery soft rotted appearance (Fig. 1), eventually became hollow and filled with sclerotia. There were no significant changes to the aboveground plant parts during the initial infection stage, but as the disease progressed, the foliage became discolored and wilted because of the damaged roots. More than 31% of the plants in a 30-ha field were infected. Symptomatic roots were collected and sclerotia were removed from the diseased tissue, immersed in 1% NaClO for 1 min, rinsed three times with sterile water, and placed on acidified potato dextrose agar (PDA) in Petri dishes. After an incubation in darkness at 20 °C for 2–3 days, 21 suspected Sclerotinia isolates were obtained. Isolates JH1 and JH2 were randomly selected for identification. On PDA, colonies produced sparse, white, and cottony aerial mycelia (i.e., wool-like appearance), with septate, branched, and hyaline hyphae. Within 4 days of incubation, the PDA surface was covered with white hyphae. Small and white sclerotial primordia formed 3 days later and were irregularly distributed in the middle and along the edge of the Petri dish. After maturing, the hardened and black sclerotia had an irregular shape and size, ranging from 1.4 × 1.5 to 4.1 × 7.5 mm (n = 50). Most of the sclerotia developed separately, with approximately 15–25 per plate (Fig. 2). On the basis of their morphology, the isolates were initially identified as Sclerotinia sp. (Mordue and Holliday 1976; Kohn 1979). Using the JH1 and JH2 rDNA internal transcribed spacer (ITS) region (GenBank accession no. MZ031405 and MZ031406) and the aspartyl protease gene specific to S. sclerotiorum (MZ292709 and MZ292710) in GenBank as queries, BLAST searches revealed that the sequences were respectively 99%–100% similar to S. sclerotiorum sequences KF859933 and AF271387. The primer pairs for amplifying the ITS region and the aspartyl protease gene were respectively ITS4/ITS5 (White et al. 1990) and SSaspr F/SSaspr R (Abd-Elmagid et al. 2013). The pathogenicity of JH1 and JH2 was evaluated using healthy plants. The roots of 4-year-old ginseng plants were washed, wiped with 75% alcohol, and transferred to flower pots containing sterile sand and sorghum grain (10:1 v/v) infested with 10-day-old isolates. For both isolates, 12 plants were inoculated, with four plants per pot. Control plants were transferred to flower pots containing sorghum grain lacking fungus. The inoculated samples were incubated in a greenhouse (12 h photoperiod and 25 °C) for 25 days before they were examined. The test was repeated twice. The inoculated roots exhibited the same symptoms as those observed in the field, whereas the controls remained symptomless. The same fungus was reisolated from all infected roots and resequencing results confirmed its identity. To the best of our knowledge, this is the first report of S. sclerotiorum causing Sclerotinia root rot on American ginseng in China. Because this disease is detrimental to the production of American ginseng, effective management strategies will need to be developed.

Highly-Expressed Aspartic Protease from Aspergillus Fumigatus-Mediated Enzymolysis of Silkworm (Bombyx Mori) Pupae Protein

Aspartic protease emerges as an optimistic hydrolytic agent to obtain several protein hydrolysates. An aspartic protease gene from Aspergillus fumigatus Af293 was successfully expressed in Pichia pastoris (GS115) and its hydrolytic potentials on silkworm (Bombyx mori) pupae protein were determined. It was optimum at pH 4.0 and 50 °C and stable over pH range 4.0-5.0 and temperatures 45-55 °C with a specific activity of 8408.9 ± 305.6 U/mg. SDS-PAGE analysis revealed the molecular weight of the recombinant protease to be 45 kDa. The half-life (t1/2) of the recombinant protease at 40, 50, 60, and 70 °C was 30, 25, 35, and 20 min, respectively. The protease showed enhanced activity in the presence of Cu2+, Pb2+ and SDS. Its substrate specificity studies were revealed in the order of cleaving ability to Bovine Serum Albumin (BSA) > Silkworm pupae powder (SPP) > Casein > Casein sodium salt (CSS). Upon hydrolysis of silkworm pupae protein, it showed enhanced and plausible hydrolytic potentials, increasing the degree of hydrolysis to 50 ± 6.1% at 6 h, increased solubility by 80%, and improved functional properties. The stable characteristics and hydrolytic performance of the recombinant aspartic protease qualify it for industrial application, especially within the food and related industries.

Single Nucleotide Polymorphisms (SNPs), Phylogeny and Spatiotemporal analysis of pro gene in HIV samples from Pakistan

Background: The increase in incidence of HIV infection with limited treatment options has enhanced the morbidity and mortality in South Asian countries. The mutations, geographical and genetic diversity in Pro gene in HIV positive patients from other South Asian countries can be helpful for treatment options. Aim: To identify mutations and genetic diversity in Pro gene in HIV positive patients in Pakistan and compare the sequences to neighbouring countries China and India. Methods: The HIV protease gene or pro-gene sequence were retrieved from NCBI. The sequence were studied from Pakistan, China and India were analysed for their linkage and genetic mutations through bioinformatics tools, MEGAX and CLUSTALW Results: The phylogenetic analysis of samples for maximum likelihood and mutation from South Asian countries Pakistan, China and India individually displayed variation in sequences of HIV pro gene in all these isolates. Whereas Pakistani isolates have more genetic similarities with the isolates from China than India. Conclusion: The phylogeny analysis depicts there is gradual evolution in viral types and possible entry is through the neighbouring transmission might have been through social connections. The mutations studies provides bases for the novel targets for the drug design and development against HIV. Keywords: HIV, Protease gene, MEGA, Phylogenetic analysis, Pakistan

First report of Sclerotinia sclerotiorum on watercress (Nasturtium officinale) in aquaponic system in Hungary

Watercress (Nasturtium officinale) is an aquatic dicotyledonous vegetable belonging to Brassicaceae (Aiton 1812). Watercress was grown in an aquaponic system on fired clay ball medium at the Aquaponic Research Station of the University of Debrecen, in the city of Debrecen (Hungary). During January 2020, 3-month-old plants showed symptoms in aquaponic cultivation. A visual survey showed 30% of plants with symptoms. Leaves and stems withered and showed white cotton-like mycelium. Mycelia from infected plants were placed on potato dextrose agar (PDA) and incubated at 25°C for seven days. Single hyphal tips were transferred to produce a pure culture. All ten fungal isolates showed similar morphological characteristics on PDA. Colonies consisted of white mycelia after three days and globoid to irregular and black 2.5 to 7 (average, 3) mm (n = 100 from ten plates) sclerotia formed ten days later, which are the typical morphological features of Sclerotinia sclerotiorum (Mordue et al. 1976). Molecular identification was performed with one of the ten isolates (Scl_B). Mycelia were grown in 250 ml of potato dextrose broth in a rotary shaker at 175 rpm at 24°C for six days. DNA was extracted from mycelium using a Nucleospin plant II (Macherey-Nagel, Germany) according to the manufacturer’s protocol. PCR amplification (Kim et al. 2014) was performed with primers ITS1/ITS4 for the internal transcribed spacer region (White et al. 1990) on a Primus 96 thermal cycler (MWG Biotech, Germany). Specific polymerase chain reaction was performed with primers SSasprF/SSasprR (Abd-Elmagid et al. 2013). PCR products were sequenced by Microsynth Austria GmbH. NCBI BLAST analysis of the 440-bp ITS sequence (Genbank MW012403.1) showed 100% identity with the sequence of S. sclerotiorum (MT177267.1, etc.). The 170-bp specific gene sequence (Genbank MW959042.1) had a 100% similarity to hypothetical proteins (Genbank MK028159.1), with a 99.4% similarity to a portion of the S. sclerotiorum aspartyl protease gene (AF271387.1). Pathogenicity tests were carried out by inoculating surface-disinfested, 30-day-old watercress plants in plastic pots (15x15x12 cm). In three repeated experiments 90 watercress plants were measured. 15 plants (one plant per pot) were planted into the five-times autoclaved substrate (Biorgmix: pH 6.1±0.5%, N:1.5%, P2O5:0.7%, K2O:0.5%, organic matter content:50%) and inoculated by ten wheat kernels that were colonized by S. sclerotiorum (Scl_B) (Garibaldi et al. 2019). 15 plants were planted into the substrate with ten non-inoculated kernels as a control. Plants were kept in an MLR-352 climatic test chamber (PHCbi, Japan) at 21 ± 1°C for 12 hr light:dark cycle. On the first day of the experiment complex nutrient solution (Tek-Land: N:5%, P2O5:5%, K2O:5%, B:0.01%, Cu:0,01%, Mn:0.02%, Mo:0.002%, Zn:0.016%) was used, then autoclaved water daily. Eight days later white mycelium appeared on every inoculated plant and five days later dark sclerotia formed on the stems. Based on the morphological characteristics the re-isolated pathogen was S. sclerotiorum. Similar results were detected in three repeated experiments with white mold fungus being reisolated from all 45 infected watercress plants. The 45 non-inoculated plants did not show any symptoms and any diseases. This pathogen has already been reported on watercress in the field (Farr et al. 1989; Boland and Hall 1994; Garibaldi et al. 2019). This is the first reported case of white mold on watercress in aquaponic system in Hungary.