Construction and functional verification of size-reduced plasmids based on TMP resistance gene dfrB10.

IMPORTANCE: Plasmid size is one of the factors affecting transfection efficacy in most of the molecular genetic research studies. One effective approach for reducing plasmid size is to replace relatively large, conventional antibiotic resistance genes with the short-size dfrB10 gene. The successful construct of a series of dfrB10-based tool plasmids and their functional validation, via comparison with original plasmids, suggest that dfrB10 is a potent drug resistance selection marker. The antibiotic trimethoprim offers convenient usage comparable to that of ampicillin or kanamycin. Additionally, fluorescence analysis has demonstrated the compatibility of TMP with protein expression in various host cells. Based on these findings, TMP-dfrB10 could be an alternative choice for future use in molecular genetic research studies that require miniature plasmids to achieve optimal results.

Active control of terahertz waves based on hybrid VO2 periodic corrugated waveguides.

We describe a method for the active control of terahertz (THz) waves using hybrid vanadium dioxide (VO2) periodic corrugated waveguide. Unlike liquid crystals, graphene and semiconductors and other active materials, VO2 exhibits a unique insulator-metal transition characteristic by the electric fields, optical, and thermal pumps, resulting in five orders of magnitude changes in its conductivity. Our waveguide consists of two gold coated plates with the VO2-embedded periodic grooves, which are placed in parallel with the grooves face to face. Simulations show that this waveguide can realize mode switching by changing the conductivity of the embedded VO2 pads, whose mechanism is attributed to the local resonance induced by defect mode. Such a VO2-embedded hybrid THz waveguide is favorable in practical applications such as THz modulators, sensors and optical switches, and provides an innovative technique for manipulating THz waves.

The Comparison of PCR Kits for the Detection of Erythrocytic Parasites on Filter Paper.

Dried blood spot (DBS) based PCR was considered an inexpensive and feasible method for detecting pathogens in the blood. The DBS carrier filter paper and PCR kits are crucial for accurate diagnosis. We evaluated 4 types of filter papers and 20 PCR kits for DBS samples. The PCR detecting Plasmodium results showed that the minimum detection limit of the 4 filter papers was 1 × 102 parasites/µL, and the positive rates of 20 PCR kits ranged from 0% to 100%. PCR results were satisfactory for detecting Plasmodium falciparum (P. falciparum) and Plasmodium. vivax (P. vivax) in archived DBS samples and Babesia gibsoni (B. gibsoni) in fresh pet DBS samples. Our results provided a useful reference for the detection of blood pathogens with DBS samples and direct PCR, especially for screening the cost-efficacy combination of filter paper and PCR kit in resource-limited areas.

The complete mitochondrial genome of major malaria vector Anopheles anthropophagus (Diptera: Culicidae) in China.

Anopheles anthropophagus (Xu and Feng 1975) is the major vector of malaria in Eastern and Southern China. The species An. anthropophagus is considered a synonym of An. lesteri (Baisas & Hu, 1936), although they differ in several key biological characteristics. Here, we report the complete mitochondrial genome of An. anthropophagus for the first time. The mitogenome of An. anthropophagus is a typical circular, double-stranded molecule with a total length of 15,413 base pairs, and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and an AT-rich control region. A phylogenetic analysis of the complete mitogenomes of 16 species of Anopheles (Culicidae) revealed that An. anthropophagus is closely related to An. sinensis (Wiedemann 1828), in the family Culicidae. The An. anthropophagus mitogenome provides new data for further taxonomic and phylogenetic studies of the genus Anopheles.

Internal Cylinder Identification Based on Different Transmission of Longitudinal and Shear Ultrasonic Waves.

We have built a Fizeau fiber interferometer to investigate the internal cylindrical defects in an aluminum plate based on laser ultrasonic techniques. The ultrasound is excited in the plate by a Q-switched Nd:YAG laser. When the ultrasonic waves interact with the internal defects, the transmitted amplitudes of longitudinal and shear waves are different. The experimental results show that the difference in transmission amplitudes can be attributed to the high frequency damping of internal cylinders. When the scanning point is close to the internal defect, the longitudinal waves attenuate significantly in the whole defect area, and their amplitude is always smaller than that of shear waves. By comparing the transmitted amplitudes of longitudinal and shear waves at different scanning points, we can achieve a C scan image of the sample to realize the visual inspection of internal defects. Our system exhibits outstanding performance in detecting internal cylinders, which could be used not only in evaluating structure cracks but also in exploring ultrasonic transmission characteristics.

Wave packet interactions in a thin aluminum plate partially immersed in water.

When investigating the wave propagation and mode conversions in a thin aluminum plate partially immersed in water, a kind of wave packet interaction was observed. It was found that the transmitted ultrasonic signal consists of different wave packets, which contain essential information of different wave types. When the incident angle is very small, the signals can be identified as the major wave packet followed by its tail. The major packet includes the information of the incident wave while the tail is related to the mode conversion and propagation in the plate. When the incident angle increased, the major packet was literally engulfed by its tail, indicating that the directly transmitted incident ultrasound disappeared and more energy was coupled into the plate. The interactions between different wave packets found here reveal the excitation and propagation mechanisms of Lamb waves in plates, which would benefit applications in ultrasonic imaging, signal recognition, underwater acoustic communication, and so on.

Actin-related protein Arp4 regulates euchromatic gene expression and development through H2A.Z deposition in blood-stage Plasmodium falciparum.

BACKGROUND: Malaria caused by Plasmodium spp. is still a major threat to public health globally. The various approaches to developing new antimalarial agents rely on the understanding of the complex regulatory mechanisms of dynamic gene expression in the life-cycle of these malaria parasites. The nuclear members of the evolutionarily conserved actin-related protein nuclear (ARP) superfamily are the major components of nucleosome remodelling complexes. In the human malaria parasite Plasmodium falciparum, bioinformatics analysis has predicted three ARP orthologues: PfArp1, PfArp4 and PfArp6. However, little is known about the biological functions of putative PfArp4. In this study, we aimed to investigate the function and the underlying mechanisms of PfArp4 gene regulation. METHODS: A conditional gene knockdown approach was adopted by incorporating the glucosamine-inducible glmS ribozyme sequence into the 3' UTR of the PfArp4 and PfArp6 genes. The transgenic parasites PfArp4-Ty1-Ribo, PfArp6-Ty1-Ribo and pL6-PfArp4-Ty1::PfArp6-HA were generated by the CRISPR-Cas9 technique. The knockdown effect in the transgenic parasite was measured by growth curve assay and western blot (WB) analysis. The direct interaction between PfArp4 and PfArp6 was validated by co-IFA and co-IP assays. The euchromatic gene expression mediated through H2A.Z (histone H2A variant) deposition and H3K9ac modification at promoters and regulated by PfArp4, was determined by RNA-seq and ChIP-seq. RESULTS: The inducible knockdown of PfArp4 inhibited blood-stage development of P. falciparum. PfArp4 and PfArp6 were colocalized in the nucleus of P. falciparum parasites. PfArp4 gene knockdown altered the global transcriptome. PfArp4 protein colocalized with the histone variant H2A.Z and euchromatic marker H3K9ac in intergenic regions. The inducible downregulation of PfArp4 resulted in the depletion of H2A.Z and lower H3K9ac levels at the upstream regions of eukaryotic genes, thereby repressing the transcriptional abundance of H2A.Z-dependent genes. CONCLUSIONS: Our findings suggest that PfArp4 regulates the cell cycle by controlling H2A.Z deposition and affecting centromere function, contributing to the understanding the complex epigenetic regulation of gene expression and the development of P. falciparum.

Esophageal Scab Mimicking a Parasite: A Case Report.

BACKGROUND Parasitic helminths in the esophagus are rare. Here, we report a case of esophageal scab mimicking a parasite. CASE REPORT A 65-year-old man was admitted to our hospital because after choking on food. Gastroscopy showed 2 foreign bodies adherent to the esophagus wall 28 and 34 cm from the incisor, which appeared to be a fluke. Two fluke-like foreign bodies (1.5 and 1.8 cm in length) were removed from the esophageal ulcer with forceps. After fixation with alcohol, the suspected fluke-like foreign bodies were noted to be brown and woody. Under a light microscope, the structure of the foreign body was not apparent, and no typical flatworm tegument structure was demonstrated on pathologic sections, but it had a blood clot-like structure. Administration of albendazole did not expel any helminths. A stool examination showed no eggs of the putative flukes. The genomic DNA of the suspected flukes was extracted and a 700 bp fragment was amplified by universal barcoding primers. The sequencing showed that the homology with human cytochrome c oxidase subunit I gene was 98.8%. CONCLUSIONS The scab formed by the esophageal ulcer was identified based on clinical manifestations, anti-helminth and stool examinations, parasite morphology, and molecular biology. Our experience with this case suggests that the universal barcoding technique can be used for identification of foreign bodies suspected to be parasites.

The use of facemasks may not lead to an increase in hand-face contact.

Advocacy of the use of facemasks by the public as a measure against the spread of COVID-19 is controversial, with some healthcare professionals arguing that the use of a face mask may increase the rate at which people touch their faces, due to readjusting the mask. We assessed the facial touching behaviour of bus passengers in China before and after the outbreak of COVID-19 and found that wearing a face mask does not increase the number of hand-face contacts and is likely, therefore, to have a positive beneficial effect on suppressing the spread of COVID-19 within populations when used in conjunction with social distancing measures.

Vanadium dioxide-assisted broadband tunable terahertz metamaterial absorber.

Tunable terahertz (THz) functional devices have exhibited superior performances due to the use of active materials, such as liquid crystals, graphene, and semiconductors. However, the tunable range of constitutive parameters of materials is still limited, which leads to the low modulation depth of THz devices. Here, we demonstrate a broadband tunable THz absorber based on hybrid vanadium dioxide (VO2) metamaterials. Unlike other phase change materials, VO2 exhibits an insulator-to-metal transition characteristic and the conductivity can be increased by 4-5 orders of magnitude under external stimulus including electric fields, optical, and thermal pumps. Based on the unique transition character of VO2, the maximum tunable range of the proposed absorber can be realized from 5% to 100% by an external thermal excitation. Meanwhile, an absorption greater than 80% in a continuous range with a bandwidth about 2.0 THz can be obtained when VO2 is in its metal phase at high temperature. Furthermore, the absorber is insensitive to the incident angle up to 50° and such a broadband THz absorber can be used in applications including imaging, modulating, cloaking, and so on.

Acoustic extraordinary transmission manipulation based on proximity effects of heterojunctions.

Heterojunctions between two crystalline semiconductor layers or regions can always lead to engineering the electronic energy bands in various devices, including transistors, solar cells, lasers, and organic electronic devices. The performance of these heterojunction devices depends crucially on the band alignments and their bending at the interfaces, which have been investigated for years according to Anderson's rule, Schottky-Mott rule, Lindhard theory, quantum capacitance, and so on. Here, we demonstrate that by engineering two different acoustic waveguides with forbidden bands, one can achieve an acoustic heterojunction with an extraordinary transmission peak arising in the middle of the former gaps. We experimentally reveal that such a transmission is spatially dependent and disappears for a special junction structure. The junction proximity effect has been realized by manipulating the acoustic impedance ratios, which have been proven to be related to the geometrical (Zak) phases of the bulk bands. Acoustic heterojunctions bring the concepts of quantum physics into the classical waves and the macroscopic scale, opening up the investigations of phononic, photonic, and microwave innovation devices.

Self-adaptive terahertz spectroscopy from atmospheric vapor based on Hilbert-Huang transform.

Absorption lines of atmospheric vapor commonly appear in terahertz (THz) spectra measured in a humid air environment. However, these effects are generally undesirable because they may mask critical spectroscopic information. Here, a self-adaptive method is demonstrated for effectively identifying and eliminating atmospheric vapor noise from THz spectra of an all-fiber THz system with the Hilbert-Huang transform. The THz signal was decomposed into eight components in different time scales called the intrinsic mode functions and the interference of atmospheric vapor was accurately isolated. A series of experiments confirmed the effectiveness and strong self-adaptiveness of the proposed system in vapor noise elimination.

Single-mode interface states in heterostructure waveguides with Bragg and non-Bragg gaps.

Interface states can always arise in heterostructures that consist of two or more (artificial) materials with topologically different energy bands. The gapped band structure can be classified by the Chern number (a topological invariant) generally or the Zak phase in one-dimensional periodic systems. Recently, topological properties have been employed to investigate the interface states occurring at the connecting regions of the heterostructures of mechanical isostatic lattices and acoustical waveguides. Here, we study this heterostructure phenomenon by carefully connecting two corrugated stainless steel waveguides with Bragg and non-Bragg gaps at approximately the same frequency. These two waveguide structures can be achieved by continuously varying their geometry parameters when a topological transition exists in the forbidden bands, in which the reflection impedance changes the sign. Furthermore, a localized single high-order mode has been observed at the interface because of the transverse mode interactions, which relate to the non-Bragg gaps created by the different transverse mode resonances. Such a localized acoustic single mode with very large enhanced intensity could find its applications in sound detection, biomedical imaging, and underwater sound control, and could also enrich our means of wave front manipulations in various engineering fields.

[Component analysis of excretory/secretory protein from Trichinella spiralis adult worm].

Objective: To analyze the component of adult worm excretory/secretory protein(AWESP) from Trichinella spiralis using the shotgun method, and find out the active component underlying its regulatory effect on colitis in humans. Methods: The T. spiralis AWESP was prepared, separated by SDS-PAGE, lysed with trypsin, and analyzed by shotgun LC-MS/MS. The protein components were determined with the Masco software and classified using the Gene Ontology(GO) method in cellular components, molecular functions, and biological processes. Results: The AWESPs isolated by SDS-PAGE had a Mr of 15 000-116 000. A total of 280 proteins were revealed by LC-MS/MS, of which 96 were identified by Masco software, 98 were putative, and the remaining 86 were unclear. Preliminary results showed that 4 proteins had regulatory potential for colitis, including cysteine protease inhibitor, serine protease, 53 000 excretory/secretory antigen, and glutathione-S-transferase. GO enrichment analysis showed that the identified proteins had 104 different molecular functions, involved in 363 biological processes. Conclusion: As revealed by the Masco software, T. spiralis AWESP has complex components and 96 have been identified in this study. Four of them are preliminarily shown to be associated with the anti-colitis effect of T. spiralis.

[Cloning, expression and identification of gametocyte specific protein Pfgdv1 of Plasmodium falciparum].

OBJECTIVE: To clone a gametocyte specific protein Pfgdv1 of Plasmodium falciparum, express and identify recombinant Pfgdvl protein in vitro. METHODS: PCR was performed to amplify Pfgdv1 from P. falciparum DNA which was got from the patient who was infected with P. falciparum, and the PCR product was inserted into pET28a (+) vector. pET28a-Pfgdv1 recombinant plasmid was constructed and transformed into E. coli host BL21 (DE3+). IPTG was used to induce the recombinant Pfgdv1 protein fused with His tag, and the protein was purified by His-NTA affinity chromatography. The recombinant protein was identified by SDS-PAGE and Western blotting. RESULTS: The PCR product of Pfgdv1 gene was about 1.65 kb, meeting the expectation of predicted fragment size. The recombinant protein was about 67 kDa, which could be recognized by His-Tag monoclonal antibody. CONCLUSION: The Pfgdv1 gene of P. falciparum is successfully cloned, and the recombinant Pfgdv1 protein is expressed, thereby providing an opportunity for further study on transmission blocking vaccine.

Vector sequence contamination of the Plasmodium vivax sequence database in PlasmoDB and In silico correction of 26 parasite sequences.

We found a 47 aa protein sequence that occurs 17 times in the Plasmodium vivax nucleotide database published on PlasmoDB. Coding sequence analysis showed multiple restriction enzyme sites within the 141 bp nucleotide sequence, and a His6 tag attached to the 3' end, suggesting cloning vector origins. Sequences with vector contamination were submitted to NCBI, and BLASTN was used to cross-examine whole-genome shotgun contigs (WGS) from four recently deposited P. vivax whole genome sequencing projects. There are at least 26 genes listed in the PlasmoDB database that incorporate this cloning vector sequence into their predicted provisional protein products.

Optimized codon usage enhances the expression and immunogenicity of DNA vaccine encoding Taenia solium oncosphere TSOL18 gene.

Cysticercosis due to larval cysts of Taenia solium, is a serious public health problem affecting humans in numerous regions worldwide. The oncospheral stage-specific TSOL18 antigen is a promising candidate for an anti-cysticercosis vaccine. It has been reported that the immunogenicity of the DNA vaccine may be enhanced through codon optimization of candidate genes. The aim of the present study was to further increase the efficacy of the cysticercosis DNA vaccine; therefore, a codon optimized recombinant expression plasmid pVAX1/TSOL18 was developed in order to enhance expression and immunogenicity of TSOL18. The gene encoding TSOL18 of Taenia solium was optimized, and the resulting opt-TSOL18 gene was amplified and expressed. The results of the present study showed that the codon-optimized TSOL18 gene was successfully expressed in CHO-K1 cells, and immunized mice vaccinated with opt-TSOL18 recombinant expression plasmids demonstrated opt­TSOL18 expression in muscle fibers, as determined by immunohistochemistry. In addition, the codon-optimized TSOL18 gene produced a significantly greater effect compared with that of TSOL18 and active spleen cells were markedly stimulated in vaccinated mice. 3H-thymidine incorporation was significantly greater in the opt-TSOL18 group compared with that of the TSOL18, pVAX and blank control groups (P<0.01). In conclusion, the eukaryotic expression vector containing the codon-optimized TSOL18 gene was successfully constructed and was confirmed to be expressed in vivo and in vitro. The expression and immunogenicity of the codon-optimized TSOL18 gene were markedly greater compared with that of the un-optimized gene. Therefore, these results may provide the basis for an optimized TSOL18 gene vaccine against cysticercosis.

[Immune response of purified Plasmodium falciparum antigen on human peripheral blood T lymphocytes in vitro].

OBJECTIVE: To explore the effects of the purified Plasmodiumfalciparum (P. f) antigen on the T lymphocytes of the peripheral blood of healthy people in vitro. METHODS: The peripheral blood mononuclear cells (PBMCs) isolated from healthy donators were cultured with purified P. fantigen (5 µg/ml) and normal RBC (nRBC) antigen (5 µg/ml), in addition, the group in which only IL-2 added was set as a negative control. After cultured for 12 d, the corresponding antigens were used to re-stimulated the activated T cells in the two stimulated groups for 20 h. Then the cells were collected, the proliferations of the T cells labeled by CFSE were analyzed, and the secretion condition of IL-4 and IFN-γ of the cells were detected by flow cytometry. RESULTS: After amplification, the proliferative index (PI) of CD4+ T cells that were stimulated with P. f antigen was significantly higher than those in the nRBC antigen group and the negative control group (both P <0.05), but there was no significant difference between the PIs of γδ T cells in the above three groups (P > 0.05). Meanwhile, the percentages of the CD4' T cells secreting IL-4+ in the P. f antigen group was significantly higher than those in the other two groups (both P <0.05), while the percentages of T cells secreting IFN-γ in the 3 groups had no significant difference (P > 0.05). CONCLUSION: The purified P.fanti- gen could effectively stimulate the proliferation of CD4+ T cells in peripheral blood of healthy people, and the latter can play a role in immunoregulation through secreting IL-4 preferentially.

Orthogonality breaking induces extraordinary single-mode transparency in an elaborate waveguide with wall corrugations.

Orthogonality plays a fundamental role in various mathematical theorems and in physics. The orthogonal eigenfunctions that represent the intrinsic motions of various physical systems can also be regarded as transverse wave modes in a straight waveguide. Because of their orthogonality, these modes propagate independently, without mutual interference. When the wall separation fluctuates, the former mode orthogonality is destroyed because of the change in the Euclidean space of the system. Here, we experimentally demonstrate the extraordinary single-mode transparency that arises as a result of the intense mode interference induced by orthogonality breaking in a waveguide with a varying cross section. A mode diagram is also introduced to illuminate these mode interactions. In particular, measurements of the transverse field distributions indicate that a three-mode interaction leads to a single high-order mode that penetrates through the lower-mode bandgaps when the wall period is carefully selected. The observation of Bessel-like transverse distributions is promising for applications in wave-control engineering.

[Construction and identification of recombinant Mycobacterium smegmatis vaccine expressing Cysticercus cellulosae cC1 antigen].

OBJECTIVE: To construct recombinant Mycobacterium smegmatis vaccine expressing Cysticercus cellulosae cC1 antigen. METHODS: The recombinant pET28a-cC1 plasmid was extracted and double digested by Xho I and BamH I restriction enzymes, and shuttle plasmid pMV261 was extracted and double digested by Hind III and BamH I restriction enzymes. Both fragments were modified by Klenow fragment to form blunt end, then the large fragments of cC1 and pMV261 plasmid were purified and ligated by T4 ligase enzyme. The recombinant pMV261-cC1 plasmid was constructed and sequenced. Then the pMV261-cC1 plasmid was transformed into Mycobacterium smegmatis by the electrotransformation method. The recombinant cC1-Mycobacterium smegmatis was induced by heat and identified by the Western blotting method with the sera of cysticercosis patients. In addition, the growth states of the Mycobacterium smegmatis and the recombinant cC1-Mycobacterium smegmatis were compared and the growth curves were drawn. RESULTS: The restriction enzyme and sequencing results showed that the recombinant pMV261-cC1 plasmid was successfully constructed. After heat induction, a 40 kD band was showed by PAGE analysis of cC1-Mycobacterium smegmatis. The Western blotting results showed that the sera of cysticercosis patients could recognize the 40 kDa band, which suggested that cC1 protein was expressed in Mycobacterium smegmatis. Compared with the Mycobacterium smegmatis, the recombi- nant cC1-Mycobacterium smegmatis showed no significant difference in proliferation characteristics. CONCLUSION: The recombinant cC1-Mycobacterium smegmatis vaccine has been successfully constructed.