Generation and Characterization of a Nanobody Against SARS-CoV

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensin-converting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

First Report of Alfalfa Anthracnose Caused by Colletotrichum americae-borealis in Xinjiang, China

Alfalfa (Medicago sativa L.) is widely planted in the world as one of the most important leguminous forage crops, and it is also the first choice of forage crops for animal feed in Xinjiang. In June 2018, alfalfa plants with typical anthracnose symptoms were observed in 75% of alfalfa fields in Hutubi County, Xinjiang, China. The disease usually occurred in alfalfa fields that had been planted for more than 2 years and was distributed in patches in the field. The incidence rate ranged from 7.5% to 53%, and the fatality rate ranged from 0 to 3%. Greater incidence was observed in fields with older stands. At the early stage of disease, pale brown prismatic or oval sunken lesions with dark brown to black edges were observed at the base of the stem of alfalfa plants. As the symptoms progressed, lesions on stems turned necrotic, and the center of the lesion became gray-white with black dots. In severe cases, the lesion expanded around the stem, causing the upper part of the stem to break off, or wilt and die. Twenty plant stem sections with typical symptoms were sampled and surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 1 min, rinsed in sterilized distilled water, dried on sterilized filter paper for 45 s, placed on potato dextrose agar (PDA), and incubated in the dark at 25°C for 7 days. A fungus was frequently isolated from the surface-sterilized segments, and the colonies of this fungus were white and flat at first, and later the center of colonies became pale brown with black microsclerotia (2.0~3.2 mm. n = 30) and white or brown acervuli (1.0~1.8 mm. n = 30). A large number of conidia and setae spread from ruptured microsclerotia under microscopy. Conidia (n = 40) were hyaline, smooth-walled, straight, aseptate, cylindrical to fusoid, both tips acute to round, 13.7 to 19.5×3.0 to 4.5 μm . Setae (n = 30) were dark brown to black, smooth-walled, 3~6 septate, straight or slightly curved, 66.9~185.1 μm long, tip round and base swollen, 3.9~5.2 μm width. Sometimes setae formed directly on hyphae or brown acervuli. Colony and conidia morphology were similar to the description of Colletotrichum americae-borealis (Damm et al. 2014; Lyu et al. 2020). DNA was extracted from fresh mycelia of three representative isolates (R11, R12 and R13) and the ITS, ACT, CHS-1 and HIS3 genes of three isolates were amplified and sequenced using the primers described previously by Damm et al. (2014). The sequences of three isolates were identical, and twelve aligned sequences from three representative isolates were deposited in GenBank (MT877442, MT877443 and MT877444 for ITS, MW854350, MW854351 and MW854352 for ACT, MW270930, MW270931 and MW270107 for CHS-1, MW854347, MW854348 and MW854349 for HIS3). Sequence analysis revealed that the ITS, ACT, CHS-1 and HIS3 sequences of three representative isolates were shared 99% (355/356 bp for HIS3) to 100% (550/550 bp for ITS, 261/261 bp for ACT, 221/221 bp for CHS-1) identities to each sequence of an American strain (CBS 136232) of C. americae-borealis from alfalfa in GenBank (NR160760 for ITS, KM105434 for ACT, KM105294 for CHS-1, KM105364 for HIS3). Four phylogenetic trees were constructed by the Mrbayes method (Damm et al. 2014), and the result showed that three representative isolates grouped with C. americae-borealis. Combined with morphological observation and molecular biological identification, the pathogen was identified as C. americae-borealis. Pathogenicity tests were executed twice on alfalfa seedlings in a greenhouse. Pots containing ten 40-day old seedlings (Xinjiang daye) were sprayed with a 100 ml of condial suspension (10^6 condia/ml) of R11. Control pots were sprayed with 100 ml of sterile distilled water. Two weeks after inoculation under greenhouse conditions (25 ± 2°C, 12-h photoperiod, 85% humidity), brown spots and necrotic lesions developed on the stem of inoculated alfalfa seedlings, which were similar to disease plants in fields, and C. americae-borealis was reisolated from symptomatic tissue. The control seedlings remained symptomless. Anthracnose caused by C. americae-borealis was reported on alfalfa in the north region of America and Iran (Damm et al. 2014; Alizadeh et al. 2015), as well as Gansu, Inner Mongolia, Yunnan and Heilongjiang Province of China ( Xu. 2019; zhang et al. 2020) . To our knowledge, this is the first report of Colletotrichum americae-borealis causing Alfalfa Anthracnose in Xinjiang, China. This finding can provide an important reference for understanding the distribution and control of this disease.

Mutualistic interaction of aphids and ants in pepper, Capsicum annuum and Capsicum frutenscens (Solanaceae)

Introduction: Adequate biological identification is fundamental for establishing integrated pest management programs and identifying the trophic and mutualist relationships that can affect pest population dynamics. Aphids are the main pest of pepper Capsicum spp. (Solanaceae) crops in Southwestern Colombia, due to their role as vectors of viruses. However, the identification of aphid species is complex, limiting the investigations performed to address their interactions with other organisms. Ants and aphids present a facultative mutualistic relationship, that promotes the growth of hemipteran colonies, for this reason, the study of the ecological mutualistic association between aphids and ants is important. Objective: The main objective was to discriminate the aphid species present in commercial crops of Capsicum spp., and to identify the ant community that attends the aphid colonies and its effects on the size of the aphid colonies. Methods: Aphid species, and their ant mutualist, were collected from Capsicum annuum and Capsicum frutescens, in the Cauca valley, Southwestern Colombia. We use the DNA barcoding approach to identify aphid species, and the ants were identified by morphology‐based taxonomy. To evaluate the effect of ant care on the size and structure of aphid colonies, generalized linear models were calculated using as the response variables the total number of aphids for each colony and the proportion of nymphs. Results: The aphid species that attack pepper crops, are: Aphis gossypii and Myzus persicae (Hemiptera: Aphididae), with A. gossypii being the species that interacts with ants (19 ant species). A. gossypii colonies attended by ants had larger sizes and more nymphs per colony, than those not attended. Conclusions: Although the aphid-ant interaction is not species-specific, it is necessary to consider its role in the propagation of viral diseases in peppers and to determine how this interaction may affect regional biological control strategies.

Current Advances in DNA Methylation Analysis Methods

DNA methylation is one of the epigenetic changes, which plays a major role in regulating gene expression and, thus, many biological processes and diseases. There are several methods for determining the methylation of DNA samples. However, selecting the most appropriate method for answering biological questions appears to be a challenging task. The primary methods in DNA methylation focused on identifying the state of methylation of the examined genes and determining the total amount of 5-methyl cytosine. The study of DNA methylation at a large scale of genomic levels became possible following the use of microarray hybridization technology. The new generation of sequencing platforms now allows the preparation of genomic maps of DNA methylation at the single-open level. This review includes the majority of methods available to date, introducing the most widely used methods, the bisulfite treatment, biological identification, and chemical cutting along with their advantages and disadvantages. The techniques are then scrutinized according to their robustness, high throughput capabilities, and cost.

Genetic differentiation of geographic populations of Rattus tanezumi based on the mitochondrial Cytb gene

Rattus tanezumi is a common domestic rat and host of the bubonic plague pathogen in China and Southeast Asia (SEA). The origin, genetic differentiation and dispersal of R. tanezumi have received increasing attention from researchers. The population genetics of R. tanezumi based on its mitochondrial cytochrome b gene have been studied to explain the origin, relationships and dispersal of populations. In this study, we captured a total of 229 rats; morphological and molecular biological identification cytochrome oxidase subunit I (COI) confirmed 131 R. tanezumi individuals collected from 6 provincial areas, and their Cytb gene sequences were analyzed. The results showed that the population in Mohan (MH), Yunnan, had the highest genetic diversity, while that in Ningde (ND), Fujian, had the lowest. Tajima’s D statistic for all populations was negative and nonsignificant, indicating the possible expansion of R. tanezumi populations. Low gene flow occurred between the Zhangmu (ZM) R. tanezumi population and other populations, and the genetic differentiation among them was high. Furthermore, our analyses revealed the ZM lineage was the oldest lineage among the groups and diverged ~1.06 Mya, followed by the Luoyang (LY) lineages (~0.51 Mya) and Yunnan lineage (~0.33 Mya). In southeastern Yunnan, the Jinshuihe (JSH) and MH populations were more closely related to the populations in southeastern China (Fuzhou (FZ), ND, Quanzhou (QZ), Nanchang (NC)) and inland areas (Chongqing (CQ), LY) than to those in other areas of Yunnan (Jiegao (JG) and Qingshuihe (QSH)), indicating that R. tanezumi may have spread from southeastern Yunnan to the interior of China. In summary, R. tanezumi may have originated in ZM and adjacent areas, spread to Yunnan, and then spread from the southeast of Yunnan inland or directly eastward from ZM to inland China.

Biomolecules and Electrochemical Tools in Chronic Non-Communicable Disease Surveillance: A Systematic Review

Over recent three decades, the electrochemical techniques have become widely used in biological identification and detection, because it presents optimum features for efficient and sensitive molecular detection of organic compounds, being able to trace quantities with a minimum of reagents and sample manipulation. Given these special features, electrochemical techniques are regularly exploited in disease diagnosis and monitoring. Specifically, amperometric electrochemical analysis has proven to be quite suitable for the detection of physiological biomarkers in monitoring health conditions, as well as toward the control of reactive oxygen species released in the course of oxidative burst during inflammatory events. Besides, electrochemical detection techniques involve a simple and swift assessment that provides a low detection-limit for most of the molecules enclosed biological fluids and related to non-transmittable morbidities.

Isolation, screening, identification and tolerance of yeast in cherry wine lees

In this study, yeast was isolated from cherry wine lees by rose Bengal medium, and its species was identified through three-stage screening, morphological observation and molecular biological identification. Moreover, the tolerance of screened strains was studied. The results showed that 30 strains of yeast were isolated from cherry wine lees, and five strains of yeast were selected, which were named YJN10, YJN16, YJN18, YJN19 and YJN28. After preliminary appraisal, strain YJN10 was Saccharomyces kudriavzevii, strain YJN16 was Saccharomyces paradoxus, and strains YJN18, YJN19, YJN28 were Saccharomyces cerevisiae. In the tolerance study, the tolerable sugar concentrations of the five strains were 650, 650, 550, 600 and 600 g/L. The tolerable alcohol volume fractions were 20, 20, 16, 18 and 18%. The tolerable molar concentration of potassium chloride was 1.8, 1.8, 1.5, 1.5 and 1.5 mol/L. Finally, strains YJN10, YJN16, YJN19 and YJN28 showed good tolerance, which laid a foundation for subsequent application in cherry wine fermentation.