Convergent genomic signatures of high-altitude adaptation among domestic mammals

Abundant and diverse domestic mammals living on the Tibetan Plateau provide useful materials for investigating adaptive evolution and genetic convergence. Here, we used 327 genomes from horses, sheep, goats, cattle, pigs and dogs living at both high and low altitudes, including 73 genomes generated for this study, to disentangle the genetic mechanisms underlying local adaptation of domestic mammals. Although molecular convergence is comparatively rare at the DNA sequence level, we found convergent signature of positive selection at the gene level, particularly the EPAS1 gene in these Tibetan domestic mammals. We also reported a potential function in response to hypoxia for the gene C10orf67, which underwent positive selection in three of the domestic mammals. Our data provide an insight into adaptive evolution of high-altitude domestic mammals, and should facilitate the search for additional novel genes involved in the hypoxia response pathway.     

Injectable hybrid inorganic nanoscaffold as rapid stem cell assembly template for cartilage repair

Cartilage injuries are often devastating and most cannot be cured because of the intrinsically low regenerative capacity of cartilage tissues. Although stem-cell therapy has shown enormous potential for cartilage repair, the therapeutic outcome has been restricted by low survival rates and poor chondrocyte differentiation in vivo. Here, we report an injectable hybrid inorganic (IHI) nanoscaffold that facilitates fast assembly, enhances survival and regulates chondrogenic differentiation of stem cells. IHI nanoscaffolds that strongly bind to extracellular matrix (ECM) proteins assemble stem cells through synergistic 3D cell–cell and cell–matrix interactions, creating a favorable physical microenvironment for stem-cell survival and differentiation in vitro and in vivo. Additionally, chondrogenic factors can be loaded into nanoscaffolds with a high capacity, which allows deep, homogenous drug delivery into assembled 3D stem-cell-derived tissues for effective control over the soluble microenvironment of stem cells. The developed IHI nanoscaffolds that assemble with stem cells are injectable. They also scavenge reactive oxygen species and timely biodegrade for proper integration into injured cartilage tissues. Implantation of stem-cell-assembled IHI nanoscaffolds into injured cartilage results in accelerated tissue regeneration and functional recovery. By establishing our IHI nanoscaffold-templated 3D stem-cell assembly method, we provide a promising approach to better overcoming the inhibitory microenvironment associated with cartilage injuries and to advance current stem-cell-based tissue engineering.     

Aberrant methylation patterns in cancer: a clinical view

Epigenetic mechanisms, such as DNA methylation, DNA hydroxymethylation, post-translational modifications (PTMs) of histone proteins affecting nucleosome remodelling, and regulation by small and large non-coding RNAs (ncRNAs) work in concert with cis and trans acting elements to drive appropriate gene expression. Advances in detection methods and development of dedicated platforms and methylation arrays resulted in an explosion of information on aberrantly methylated sequences linking deviations in epigenetic landscape with the initiation and progression of complex diseases. Here, we consider how DNA methylation changes in malignancies, such as breast, pancreatic, colorectal, and gastric cancer could be exploited for the purpose of developing specific diagnostic tools. DNA methylation changes can be applicable as biomarkers for detection of malignant disease in easily accessible tissues. Methylation signatures are already proving to be an important marker for determination of drug sensitivity. Even more, promoter methylation patterns of some genes, such as MGMT, SHOX2, and SEPT9, have already been translated into commercial clinical assays aiding in patient assessment as adjunct diagnostic tools. In conclusion, the changes in DNA methylation patterns in tumour cells are slowly gaining entrance into routine diagnostic tests as promising biomarkers and as potential therapeutic targets.Key words: biomarkers, CpG islands, DNA methylation, molecular diagnostics, epigenetics     

Efficiency of RAPD,ISSR, AFLP and ISTR markers for the detection of polymorphisms and genetic relationships in camote de cerro (Dioscorea spp.)

BackgroundAt present, species known as camote de cerro (Dioscorea spp.) are found only in the wilderness in Mexico, but their populations are extremely depleted because they are indiscriminately collected, it is urgent to evaluate the conservation status of these plants in order to design conservation genetics programs. In this study, genetic diversity parameters along with cluster analysis based on Jaccard's coefficient were estimated with the objective to assess the efficiency of Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR), Amplified Fragment Length Polymorphism (AFLP) and Inverse Sequence Tagged Repeat (ISTR) molecular DNA markers in the Dioscorea genus.ResultsThe polymorphic information contents were quite similar for all markers (≈ 0.48). Genetic variation of Dioscorea spp., in terms of average heterozygosity was lower with ISTR (0.36), and higher when other markers were used (RAPD = 0.43; ISSR = 0.45 and AFLP = 0.47).ConclusionThis indicates an important level of genetic differences despite the fact that the plant is asexually propagated. Based on the diversity statistics, any marker tested in present work can be recommended for use in large-scale genetic studies of populations. However, the low correlations among different molecular marker systems show the importance of the complementarity of the information that is generated by different markers for genetic studies involving estimation of polymorphism and relationships.     

Oral Nicotine Induces Oxidative Stress and Inflammation but Does Not Subvert Tumor Suppressor and DNA Repair Responses in Mice

Nicotine, responsible for the addictive properties of tobacco, is widely used in nicotine replacement therapy for tobacco use cessation. We investigated the time-dependent effect of treatment with nicotine on the tumor suppressor, DNA repair and immune responses. Swiss Albino mice (laca strain) of both sexes received nicotine dissolved at a dose of 100 µg/ml in 2% sucrose for 24 weeks, by oral gavage, while age- and gender-matched controls received only 2% sucrose for the same period. Nicotine-treated and control mice were sacrificed 6, 16 and 24 weeks post-treatment, and their tissues evaluated for alterations in histology, oxidative stress, TNF-α levels, nitric oxide (NO) and myeloperoxidase (MPO) release, tumor suppressor response and DNA repair response. Statistical significance of results was determined using Students’ t test. The tissues of nicotine treated mice exhibited a large number of multinucleated and binucleated cells, enlarged nuclei and non-uniform distribution of cells, significant increase in expression of TNF-α gene and serum TNF-α, and time-dependent significant increase in lipid peroxidation, protein carbonylation, NO and MPO release when compared to age-and gender-matched controls. The mRNA expression of the tumor suppressor gene p53, its primary regulator Mdm2, and the DNA repair genes Brca2 and Ape1 were significantly elevated, but the corresponding protein levels remained largely unaltered. In conclusion, treatment with nicotine caused oxidative stress and inflammation which can cause widespread cellular damage from the very onset of treatment, without subverting the tumor suppressor and DNA repair responses.Electronic supplementary materialThe online version of this article (10.1007/s12291-020-00903-8) contains supplementary material, which is available to authorized users.     

EGFR Mutation Analysis in Non-small Cell Lung Carcinoma Patients: A Liquid Biopsy Approach

In the era of the targeted therapy identification of EGFR mutation detection in lung cancer is extremely helpful to predict the treatment efficacy of EGFR tyrosine kinase inhibitors (TKIs). Unfortunately, the inadequacy and quality of the biopsy samples are the major obstacles in molecular testing of EGFR mutation in lung cancer. To address this issue, the present study intended to use liquid biopsy as the non-invasive method for EGFR mutation detection. A total of 31 patients with an advanced stage of lung cancer were enrolled in the study from which cell-free DNA (cfDNA) and FFPE tissue DNA was extracted. Extracted DNA samples were analyzed for further EGFR exon specific mutation analysis by ARMS-PCR. Data were analyzed statistically using SPSS software. In cfDNA samples, the prevalence of wild type EGFR was 48% while the prevalence of TKI resistant and TKI sensitive mutations were 3%. Conversely, in tissue DNA samples, the prevalence of wild type, TKI sensitive and TKI resistant mutations were 48%, 19%, and 3%, respectively. The overall concordance of EGFR mutation between cfDNA and tissue DNA was 83%. McNemar’s test revealed that there was no significant difference between EGFR expression of cfDNA and tissue DNA samples. Additionally, the significant-high incidence of TKI resistant mutations was observed in tobacco habituates, indicating the role of carcinogens present in the tobacco in developing resistant mutations. In conclusion, our data suggest that evaluation of EGFR mutation from cfDNA samples is practicable as a non-invasive tool in patients with advanced-stage of lung cancer.     

Single nucleotide polymorphisms in partial sequences of the gene encoding the large sub-units of ADP-glucose pyrophosphorylase within a representative collection of 10 Musa genotypes

BackgroundADP-glucose pyrophosphorylase (AGPase) is a rate-limiting enzyme catalyzing the first step in the starch biosynthesis pathway in higher plants. To date, there are no reported variants or isoforms of the AGPase enzyme in bananas (Musa spp. family Musaceae) as is the case of other plants. In this study, genomic DNA sequences homologous to the gene encoding one of the large subunits of the enzyme were amplified from 10 accessions of the genus Musa, including representatives of wild ancestors (AA and BB genomes), dessert bananas (AA, AAA, AB and AAB genomes), plantains (AAB genome) and cooking bananas (ABB and AAA genomes), and studied in order to find single nucleotide polymorphisms (SNP) base variations in Musa accessions.ResultsIn the 810-base pair amplicons of the AGPase large sub-unit (LSU) gene analyzed in ten Musa accessions, a total of 36 SNPs and insertions/deletions (indels) were found. The phylogenetic analysis revealed fifteen distinct haplotypes, which were grouped into four variants. Deep examination of SNPs in the 2nd exon in the LSU of AGPase showed that at seven locations, five SNPs altered their amino acid sequence.ConclusionsThis work reveals the possible number of AGPase enzyme isoforms and their molecular levels in banana. Molecular markers could be designed from SNPs present in these banana accessions. This information could be useful for the development of SNP-based molecular markers for Musa germplasm, and alteration of the allosteric properties of AGPase to increase the starch content and manipulate the starch quality of banana fruits.     

Social movements and institutional entrepreneurship as facilitators of technology transition: The case of free/open-source software

We integrate insights from the literature on social movements and institutional entrepreneurship into the strategic niche management (SNM) and multilevel perspective (MLP) frameworks to understand the emergence of Linux, a free/open-source operating system, in a regime dominated by proprietary operating systems such as Unix and Windows NT. Employing a “microhistories” methodology, we document how actors in the free/open-source movement took steps that enabled an alternate technological niche to form, gain momentum and eventually infiltrate the extant regime. Our account delineates the key role that actors play in shaping the identity of a niche, amplifying its presence, and finally mainstreaming it. We observe a heterogenous response by incumbents to the emergent niche and highlight the sustained coexistence of a niche and regime as a distinct form of technological transition. Finally, we demonstrate the significant impact that a niche can have, spanning beyond the targeted regime, and becoming part of the landscape. Our insights highlight how tracing the processes involved in the emergence and development of a niche can provide a prospective and generative understanding of technological transition, thereby contributing to and complementing the extant SNM and MLP literatures.     

Vibrio sp. ArtGut-C1, a polyhydroxybutyrate producer isolated from the gut of the aquaculture live diet Artemia (Crustacea)

BackgroundVibrio species display variable and plastic fitness strategies to survive and interact with multiple hosts, including marine aquaculture species that are severely affected by pathogenic Vibrios. The culturable Vibrio sp. strain ArtGut-C1, the focus of this study, provides new evidence of such phenotypic plasticity as it accumulates polyhydroxybutyrate (PHB), a biodegradable polymer with anti-pathogen activity, particularly in the marine larviculture phase. The strain was isolated from the gut of laboratory-reared Artemia individuals, the live diet and PHB carrier used in larviculture. Its main phenotypic properties, taxonomic status and genomic properties are reported based on the whole-genome sequencing.ResultsVibrio sp. ArtGut-C1 yielded 72.6% PHB of cells’ dry weight at 25°C. The genomic average nucleotide identity (ANI) shows it is closely related to V. diabolicus (ANI: 88.6%). Its genome contains 5,236,997-bp with 44.8% GC content, 3,710 protein-coding sequences, 96 RNA, 9 PHB genes functionally related to PHB metabolic pathways, and several genes linked to competing and colonizing abilities.ConclusionsThis culturable PHB-accumulating Vibrio strain shows high genomic and phenotypic variability. It may be used as a natural pathogen biocontrol in the marine hatchery and as a potential cell factory for PHB production.How to cite: Yévenes M, Quiroz M, Maruyama F, et al. Vibrio sp. ArtGut-C1, a polyhydroxybutyrate producer isolated from the gut of the aquaculture live diet Artemia (Crustacea). Electron J Biotechnol 2021;49. https://doi.org/10.1016/j.ejbt.2020.10.003     

Francisco Sánchez and the Quaestio de certitudine mathematicarum: A sceptical approach

In this paper I analyse Francisco Sánchez’s role in the Quaestio de certitudine mathematicarum debate. Despite some studies on the philosophical and medical scepticism of Sánchez and, his extant letter with Christopher Clavius, a participant in the debate, we have few analyses about Sánchez’s position regarding the certainty of mathematics. Sánchez discussed some problems that Clavius analysed in his Prolegomena to propose an empirical basis for mathematics through a questioning of its certainty. I will trace the conceptual connections between Sánchez’s 1589 letter to Clavius and the Quaestio debate, to introduce Sánchez’s sceptical approach to analysing the certainty of mathematics.     

Escherichia coli GutM4 produces 2,5-diketopiperazines and inhibits human pathogens in vitro

BackgroundIt has been a very common practice to use probiotics or their metabolites as alternative antimicrobial strategies for the treatment and prevention of infections as rampant and indiscriminate use of antibiotics causes the development of antibiotic-resistant pathogens. The objective of this study was to select a potential antimicrobial probiotic strain of Escherichia coli from the human gastrointestinal tract and investigate the production of diketopiperazines that contribute to the antimicrobial activity.ResultsE. coli GutM4 was isolated from the feces of a healthy adult. E. coli GutM4 showed significant antagonistic activity against 10 indicator pathogens, and this activity was no less than that of the reference strain E. coli Nissle 1917 against eight of the indicator pathogens. Moreover, E. coli GutM4 produced antagonistic substances containing trypsin-targeted peptide bonds because the inhibitory effects of E. coli GutM4 supernatant significantly decreased upon treatment with trypsin. Consistent with the antagonistic activity and peptide compounds of E. coli GutM4, 14 2,5-diketopiperazines were isolated from the fermented broth of E. coli GutM4, including 12 cyclo(Pro-Phe), 3 cyclo(Pro-Tyr), and 5 cyclo(4-hydroxyl-Pro-Leu), which are reported to have antipathogenic activity.ConclusionE. coli GutM4 produces 2,5-diketopiperazines that are partly involved in antagonistic action against human pathogens in vitro.     

Development of a new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria

BackgroundFreeze-drying is known as one of the best methods to preserve bacterial strains. Protectant is the key factor affecting the survival rate of freeze-dried strains. In addition, salinity, bacterial suspension concentration, drying time, and other factors can also affect the survival rate of strains to varying degrees. At present, there are relatively few studies on freeze-drying preservation of marine bacteria. In the present study, we performed the freeze-drying protectant screening and optimized the preservation conditions for Pseudoalteromonas nigrifaciens, which is widely distributed in marine environment. The protective effects of the screened protectants were verified by 18 other marine bacterial strains.ResultsThe results indicated that the combination of 5.0% (w/v) lactose, 5.0% (w/v) mannitol, 5.0% (w/v) trehalose, 10.0% (w/v) skim milk powder, 0.5% (w/v) ascorbic acid and 0.5% (w/v) gelatin was the best choice for the preservation of P. nigrifaciens. The suggested salinity and concentration of initial cell suspension were 10 g/L NaCl and 1.0 × 10⁹ CFU/mL, respectively. Furthermore, stationary-phase cells were the best choice for the freeze-drying process. The highest survival rate of P. nigrifaciens reached 52.8% when using 5–10% (w/v) skim milk as rehydration medium. Moreover, the other 18 marine strains belonging to Pseudoalteromonas, Vibrio, Photobacterium, Planomicrobium, Edwardsiella, Enterococcus, Bacillus, and Saccharomyces were freeze-dried under the abovementioned conditions. Their survival rates were 2.3–95.1%.ConclusionCollectively, our results supported that the protectant mixture and parameters were beneficial for lyophilization of marine bacteria.How to cite: Zhang Z, Yu Y, Wang Y, et al. Development of new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria. Electron J Biotechnol 2020;44. https://doi.org/10.1016/j.ejbt.2019.12.006.     

High genetic diversity and small genetic variation among populations of Magnolia wufengensis (Magnoliaceae), revealed by ISSR and SRAP markers

BackgroundGenetic diversity and genetic variation of 10 populations and subpopulations of Magnolia wufengensis, a new and endangered endemic species, were examined by inter simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) molecular markers. Compared with other endangered endemic Magnolia taxa, M. wufengensis holds a relatively high level of genetic variation.ResultTotal genetic diversity was found to be 87.7% for ISSR and 88.0% for SRAP markers. For polymorphic loci (P), the effective mean number of alleles (Ae) was 1.414 for ISSR markers and 1.458 for SRAP markers, while the mean expected heterozygosity (H) was 0.256 using ISSR and 0.291 for SRAP markers. Within-population variation was estimated for P as 74.9% using ISSR and 74.6% with SRAP markers; the number of alleles Ae was 1.379 with ISSR and 1.397 for SRAP and H 0.235 with ISSR and 0.247 for SRAP markers.ConclusionThe analysis of molecular variation of both ISSR and SRAP marker systems indicated that most genetic variation is within populations, with values of 90.64% and 82.92% respectively. Mantel tests indicated a moderate association between the two marker systems and a low correlation between genetic and geographic distances. High levels of genetic diversity and low levels of population divergence suggest that genetic drift is not currently of great concern for this species. Severe habitat loss and fragmentation, predominantly ascribed to anthropogenic pressures, caused in-situ developing restriction of this species. Action for conserving this rare species for its long-term survival should be taken immediately.     

Isolation of cells for selective treatment and analysis using a magnetic microfluidic chip

This study describes the development and testing of a magnetic microfluidic chip (MMC) for trapping and isolating cells tagged with superparamagnetic beads (SPBs) in a microfluidic environment for selective treatment and analysis. The trapping and isolation are done in two separate steps; first, the trapping of the tagged cells in a main channel is achieved by soft ferromagnetic disks and second, the transportation of the cells into side chambers for isolation is executed by tapered conductive paths made of Gold (Au). Numerical simulations were performed to analyze the magnetic flux and force distributions of the disks and conducting paths, for trapping and transporting SPBs. The MMC was fabricated using standard microfabrication processes. Experiments were performed with E. coli (K12 strand) tagged with 2.8 μm SPBs. The results showed that E. coli can be separated from a sample solution by trapping them at the disk sites, and then isolated into chambers by transporting them along the tapered conducting paths. Once the E. coli was trapped inside the side chambers, two selective treatments were performed. In one chamber, a solution with minimal nutrition content was added and, in another chamber, a solution with essential nutrition was added. The results showed that the growth of bacteria cultured in the second chamber containing nutrient was significantly higher, demonstrating that the E. coli was not affected by the magnetically driven transportation and the feasibility of performing different treatments on selectively isolated cells on a single microfluidic platform.     

Enantioselective assembly of multi-layer 3D chirality

The first enantioselective assembly of sandwich-shaped organo molecules has been achieved by conducting dual asymmetric Suzuki-Miyaura couplings and nine other reactions. This work also presents the first fully C-C anchored multi-layer 3D chirality with optically pure enantiomers. As confirmed by X-ray diffraction analysis that this chiral framework is featured by a unique C₂-symmetry in which a nearly parallel fashion consisting of three layers: top, middle and bottom aromatic rings. Unlike the documented planar or axial chirality, the present chirality shows its top and bottom layers restrict each other from free rotation, i.e., this multi-layer 3D chirality would not exist if either top or bottom layer is removed. Nearly all multi-layered compounds showed strong luminescence of different colors under UV irradiation, and several randomly selected samples displayed aggregation-induced emission (AIE) properties. This work is believed to have broad impacts on chemical, medicinal and material sciences including optoelectronic materials in future.     

Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris

BackgroundHuman is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to speed up the search for more cost-effective and target specific hTopoI inhibitors is warranted. This study aims to compare the optimised conditions for the expression of hTopoI in KM71H (Mut^S) and X33 (Mut⁺) strains of Pichia pastoris. P. pastoris transfected with an hTopoI recombinant vector was used for the optimization of a higher level of hTopoI expression.ResultsIn the process, fed-batch cultivation parameters that influence the expression of hTopoI, such as culture temperature, methanol induction and feeding strategy, were optimised in the transfected KM71H and X33 P. pastoris strains in a shake flask system. The cell density and total protein concentration (protein level) of transfected P. pastoris were compared to determine the optimum culture conditions for each transfected P. pastoris strain. A higher hTopoI level was observed in the transfected KM71H culture supernatant (2.26 ng/mL) when the culture was incubated in the optimum conditions.ConclusionsThis study demonstrated that Mut^S strain (KM71H) expressed and secreted a higher level of hTopoI heterologous protein in the presence of methanol compared to the Mut⁺ strain; X33 (0.75 ng/mL). However, other aspects of optimization, such as pH, should also be considered in the future, to obtain the optimum expression level of hTopoI in P. pastoris.     

Rapid detection of live methicillin-resistant Staphylococcus aureus by using an integrated microfluidic system capable of ethidium monoazide pre-treatment and molecular diagnosis

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium resistant to all existing penicillin and lactam-based antimicrobial drugs and, therefore, has become one of the most prevalent antibiotic-resistant pathogens found in hospitals. The multi-drug resistant characteristics of MRSA make it challenging to clinically treat infected patients. Therefore, early diagnosis of MRSA has become a public-health priority worldwide. Conventionally, cell-culture based methodology and microscopic identification are commonly used for MRSA detection. However, they are relatively time-consuming and labor-intensive. Recently, molecular diagnosis based on nucleic acid amplification techniques, such as polymerase chain reaction (PCR), has been widely investigated for the rapid detection of MRSA. However, genomic DNA of both live and dead pathogens can be distinguished by conventional PCR. These results thus could not provide sufficient confirmation of an active infection for clinicians. In this study, live MRSA was rapidly detected by using a new integrated microfluidic system. The microfluidic system has been demonstrated to have 100% specificity to detect live MRSA with S. aureus and other pathogens commonly found in hospitals. The experimental results showed that the limit of detection for live MRSA from biosamples was approximately 10² CFU/μl. In addition, the entire diagnostic protocol, from sample pre-treatment to fluorescence observation, can be automatically completed within 2.5 h. Consequently, this microfluidic system may be a powerful tool for the rapid molecular diagnosis of live MRSA.     

Focused ion beam milling of microchannels in lithium niobate

We present experimental and simulation results for focused ion beam (FIB) milling of microchannels in lithium niobate in this paper. We investigate two different cuts of lithium niobate, Y- and Z-cuts, and observe that the experimental material removal rate in the FIB for both Y-cut and Z-cut samples was 0.3 μm³/nC, roughly two times greater than the material removal rate previously reported in the literature but in good agreement with the value we obtain from stopping and range of ions in matter (SRIM) simulations. Further, we investigate the FIB milling rate and resultant cross-sectional profile of microchannels at various ion beam currents and find that the milling rate decreases as a function of ion dose and correspondingly, the cross-sectional profiles change from rectangular to V-shaped. This indicates that material redeposition plays an important role at high ion dose or equivalently, high aspect ratio. We find that the experimental material removal rate decreases as a function of aspect ratio of the milled structures, in good agreement with our simulation results at low aspect ratio and in good agreement with the material removal rates previously reported in the literature at high aspect ratios. Our results show that it is indeed easier than previously assumed to fabricate nanochannels with low aspect ratio directly on lithium niobate using the FIB milling technique.