Molecular Identification of Hyalomma Ticks and Application of Bacillus thuringiensis Toxins as an Effective Biological Acaricide.

Bacillus thuringiensis (B. thuringiensis) is considered one of the most important entomopathogenic microorganisms. It produces potent toxins against insects. Therefore, the present study investigates the bioacaricidal properties of B. thuringiensis on the Hyalomma tick species. Firstly, we identify Hyalomma ticks based on morphological screening and molecular characterization. The cytochrome C oxidase subunit I (COX1) gene was selected for the polymerase chain reaction (PCR) analysis, which resulted in the amplification of 656 bp. The amplified products were sequenced, and the isolated (COX1) gene of ticks was submitted to the gene bank of NCBI (Accession No. OR077934.1). The nucleotide sequences were retrieved from the NCBI data bank by BLASTn analysis, which confirmed that all obtained sequences belong to genus Hyalomma, and multiple alignments confirmed that the sequence of Hyalomma anatolicum Tandojam-isolate (HA-TJ) 100% aligned with Hyalomma analoticum KP792577.1, Hyalomma detritum KP792595.1, Hyalomma excavatum KX911989.1, and H. excavatum OQ449693.1. The generated phylogenetic tree confirmed that sequences of HA-TJ COX1 clustered with a single clad of H. analoticum, H. excavatum, and H. detritum. The acaricidal effect of B. thuringiensis toxins B. thuringiensis spore crystal mix (BtSCM) and B. thuringiensis crystal proteins (Btcps) was evaluated against larvae and adult life stages of Hyalomma ticks in vitro. We applied Btcps and BtSCM separately with different concentrations and calculated the mortality percentage. Adult mortality was estimated at the 8th, 10th, 12th, and 15th days posttreatment and larval mortality after 24 h. During treatment of the adult life stage, at first, ticks were immersed in different concentrations of Btcps and BtSCM for 5 min after the treatments, and the samples were transferred to sterile containers and placed in an incubator with 80% humidity at 23°C. Furthermore, Btcps produced the highest mortality on Day 15, 89 ± 1.00% at a concentration of 3000 µg/mL, followed by the 12th, 10th, and 8th days produced 83 ± 1.91%, 70 ± 1.15%, and 61 ± 1.00%, respectively. BtSCM produced mortality of 69 ± 1.91% on Day 15 at a concentration of 3000 µg/mL, followed by the 12th, 10th, and 8th days at 57 ± 2.51%, 37 ± 1.91%, and 34 ± 2.00%. The present study revealed that B. thuringiensis toxins produced a significant (p < 0.05) increase in mortality rate in adults of Hyalomma ticks. Additionally, Btcps and BtSCM were used to treat the larval stage. The treatments were applied to calculate the mortality percentage via the Laravel packet test. At a 1500 µg/mL concentration, Btcps resulted in the highest mortality of 98 ± 1.15%; this was followed by 1250 µg/mL, 1000 µg/mL, and 750 µg/mL, which produced mortalities of 76 ± 1.63%, 60 ± 1.63%, and 56 ± 1.63%, respectively. In addition, BtSCM produced a mortality rate of 79 ± 2.51% at a concentration of 1500 µg/mL. Furthermore, 75 ± 2.51%, 65 ± 1.91%, and 58 ± 1.15% mortality were observed at concentrations of 1250 µg/mL, 1000 µg/mL, and 750 µg/mL, respectively. The results showed a significant (p < 0.05) increase in larval mortality compared to the control group. We conclude that B. thuringiensis toxins are applicable as a bioacaricide.

Cellular activity of autophagy and multivesicular bodies in lens fiber cells during early lens development in rbm24a mutant of zebrafish: Ultrastructure analysis.

Use of zebrafish as animal model for various diseases during early developmental stages has been exponentially increased with the aim to achieve the best representative results in this transparent fish. Recent studies documented that Rbm24a mutant causes cataract formation and resulted in blindness using the zebrafish model. Therefore, correct interpretation of studies that aimed for molecular approaches, a description of comparative and in-depth analysis of development of lens in wildtype and mutant is crucial to obtain the correct conclusion. In this study, we use a gold standard method the Transmission Electron Microscopy (TEM) to analysis the lens development in rbm24a mutant zebrafish. Firstly, we compare the cellular structures at 16-20 h post fertilization (hpf), the lens placode in ectoderm indicated delay lens development in rbm24a mutant than wildtype (siblings) zebrafish. At 33 hpf, loosely appeared lens fiber cells showed heterogenous electron density with numbers of mitochondria in lens of rbm24a mutant, revealed the influence of gene mutation in lens development. A detail ultrastructure of lens of rbm24a mutant also presented at 33 hpf. Comparatively in wildtype (siblings) at 33 hpf, lens exhibited homogenous electron density in tightly packed lens fiber cells with few mitochondria. Furthermore, to characterize the lens in rbm24a mutant we obtained data of cellular structures on 25 hpf and 1.5 days' post fertilization (dpf). At 25 hpf in mutant zebrafish, the detached solid sphere lens mass from ectoderm showed karyorrhexis, mitophagy and vesicles (also multivesicular bodies), these cellular structures supposed to hamper the development of future fiber cells. Moreover, at 1.5 dpf in mutant, nuclear excisosome, multilamellar bodies and irregular shaped mitochondria in heterogenous electron dense cytoplasm of lens fiber cells, collectively shown affected lens transparency. In summary the ultrastructure results of lens of rbm24a mutant zebrafish expand our knowledge and give reflection of different cellular activities like autophagy, apoptosis, vesicles (multivesicular bodies) and nuclear excisosomes which play their role in transparency achievement.

Interaction of Epididymal Epithelia and their Secretions with Spermatozoa Supports Functional and Morphological Changes During Long-Term Storage in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

Post-testicular maturation of spermatozoa is crucial for attaining the morphological and functional capabilities needed for successful fertilization. Epididymal epithelia offer a favorable environment for spermatozoa that are stored long term in the turtle epididymis; however, sperm-epithelial interactions during storage, which are enormously important for sperm functional and morphological maturation, are still largely unknown in turtles. The present study examined the epididymis during the sperm-storage period (November-April) in the Chinese soft-shelled turtle (Pelodiscus sinensis). Light and transmission electron microscopy were used to determine the cellular features of each epididymal segment (caput, corpus, and cauda) and their epithelial interactions with the spermatozoa. Spermatozoa were mainly located in the lumena of caput, corpus, and cauda epididymides. Numerous spermatozoa were bound to apical surfaces of the epithelia, and several were even embedded in the epithelial cytoplasm of the caput and corpus epididymides. No embedded spermatozoa were found in the cauda epididymis. In all epididymal segments, principal and clear cells showed the synthetic activity, evidenced by a well-developed endoplasmic reticulum network and high and low electron-dense secretory materials, respectively. Principal and clear cells in the caput and corpus segments showed embedded spermatozoa in electron-dense secretions and in the lipid droplets within the cytoplasm. No lysosomes were observed around the embedded spermatozoa. The lumena of the caput and corpus segments showed few apocrine and low electron density secretions. In the lumen of the cauda epididymidis, different secretions, such as holocrine with low and high electron density and their fragmentation, apocrine, and dictyosome, were found and are summarized. Altogether, sperm physical interactions with secretions either in the cytoplasm of epithelium or in the lumen may support the viability, morphological maintenance, and transfer of various proteins involved in long-term sperm storage in the turtle. This interaction could help us to understand the mechanisms of long-term sperm storage and provide more insights into the reproductive strategies of turtle sperm preservation.

Effects of topical Vancomycin Dressing on Methicillin-Resistant Staphylococcus Aureus (MRSA) positive diabetic foot ulcers.

OBJECTIVE: To compare the effects of simple saline dressings versus topical vancomycin dressings on Methicillin-resistant Staphylococcus Aureus positive chronic diabetic foot ulcers. METHODS: It was quasi experimental study conducted in Combined Military Hospital Kohat and PNS-Shifa Hospital Karachi from 01 January 2017 to 31 December 2017. A total of 23 patients were included based on non-probability convenient sampling who had diabetes and presented with foot ulcers for more than two weeks showing positive growth of Methicillin-Resistant Staphylococcus Aureus. The patients were treated with simple saline soaked dressings and debridement at first for three weeks followed by three weeks of topical vancomycin dressings with debridement. Thus patients served as their own controls. RESULTS: The average change in surface area with saline dressing was +1.73 ±1.53cm2 per week whereas with vancomycin soaked dressing it was --0.06±1.60 cm2 per week (p <0.05). The average exudate also decreased from 1.78±1.23 to 0.99±0.72 (p<0.05) and same trend was observed in percentage of slough covering the ulcer from 45% ± 22.3% to 24.3% ±12.90% (p<0.05) with vancomycin dressing. Moreover, fifteen patients had negative culture for MRSA within 2 weeks. CONCLUSION: Vancomycin impregnated dressing in MRSA positive Diabetic foot may help achieve early healing as compared to simple conventional dressings with no systemic toxicity.

Crystal structure of zebrafish complement 1qA globular domain.

C1q contains three globular domains (C1qgD) that are the key functional component of the classical complement system. C1qgD can interact with important immune molecules, including IgG and C-reactive protein (CRP) to form defense systems to protect animals. Here, the first non-mammalian structure, zebrafish C1qA globular domain (Dare-C1qAgD) was solved. Although the overall architecture of Dare-C1qAgD is similar to human C1qA, residues involved in C1qBgD, C1qCgD, and CRP binding are somewhat different while residues involved in IgG binding are not present in zebrafish. The structure gives insight into how human and fish C1qA evolved from an ancestral protein.

Crystallization and preliminary X-ray diffraction analysis of the two distinct types of zebrafish ß2-microglobulin.

ß(2)-Microglobulin (ß(2)m) noncovalently associates with the heavy chain of major histocompatibility complex class I (MHC I) molecules, which bind foreign antigen peptides to control the cytotoxic T lymphocyte (CTL) immune response. In contrast to mammals, there are distinct types of ß(2)ms derived from two loci in a number of teleost species. In order to clarify the structures of the ß(2)ms, the zebrafish (Danio rerio) ß(2)ms Dare-ß(2)m-I and Dare-ß(2)m-II were expressed in Escherichia coli, purified and crystallized, and diffraction data were collected to 1.6 and 1.9 Å resolution, respectively. Both crystals belonged to space group P2(1)2(1)2(1). The unit-cell parameters were determined to be a = 38.2, b = 50.4, c = 50.9 Å for Dare-ß(2)m-I and a = 38.9, b = 52.7, c = 65.8 Å for Dare-ß(2)m-II. Each asymmetric unit was constituted of one molecule, with Matthews coefficients of 2.22 and 3.01 Å(3) Da(-1) and solvent contents of 45 and 59% for Dare-ß(2)m-I and Dare-ß(2)m-II, respectively. These two ß(2)m structures will provide relevant information for further studies of the structures of the MHC I complex.

De novo transcriptomic analysis of peripheral blood lymphocytes from the Chinese goose: gene discovery and immune system pathway description.

BACKGROUND: The Chinese goose is one of the most economically important poultry birds and is a natural reservoir for many avian viruses. However, the nature and regulation of the innate and adaptive immune systems of this waterfowl species are not completely understood due to limited information on the goose genome. Recently, transcriptome sequencing technology was applied in the genomic studies focused on novel gene discovery. Thus, this study described the transcriptome of the goose peripheral blood lymphocytes to identify immunity relevant genes. PRINCIPAL FINDINGS: De novo transcriptome assembly of the goose peripheral blood lymphocytes was sequenced by Illumina-Solexa technology. In total, 211,198 unigenes were assembled from the 69.36 million cleaned reads. The average length, N50 size and the maximum length of the assembled unigenes were 687 bp, 1,298 bp and 18,992 bp, respectively. A total of 36,854 unigenes showed similarity by BLAST search against the NCBI non-redundant (Nr) protein database. For functional classification, 163,161 unigenes were comprised of three Gene Ontology (Go) categories and 67 subcategories. A total of 15,334 unigenes were annotated into 25 eukaryotic orthologous groups (KOGs) categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) database annotated 39,585 unigenes into six biological functional groups and 308 pathways. Among the 2,757 unigenes that participated in the 15 immune system KEGG pathways, 125 of the most important immune relevant genes were summarized and analyzed by STRING analysis to identify gene interactions and relationships. Moreover, 10 genes were confirmed by PCR and analyzed. Of these 125 unigenes, 109 unigenes, approximately 87%, were not previously identified in the goose. CONCLUSION: This de novo transcriptome analysis could provide important Chinese goose sequence information and highlights the value of new gene discovery, pathways investigation and immune system gene identification, and comparison with other avian species as useful tools to understand the goose immune system.

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the chicken CD8αα-BF2*0401 complex.

In the process of antigen presentation, the MHCI-CD8 complex is important for immune signal transduction by the activation of cytotoxic T cells. Here, the expression, purification, crystallization and X-ray analysis of the complex of the chicken MHC class I molecule BF2*0401 and CD8αα (CD8αα-BF2*0401) are reported. This complex was verified by SDS-PAGE analysis of a CD8αα-BF2*0401 crystal, which showed three bands corresponding to the molecular weights of BF2*0401, ß2-microglobulin and CD8α, respectively. The crystal of CD8αα-BF2*0401 diffracted to 2.8 Šresolution and belonged to space group P21, with unit-cell parameters a = 90.6, b = 90.8, c = 94.9 Å, ß = 98°. The Matthews coefficient and solvent content were calculated to be 2.88 Å(3) Da(-1) and ∼57.3%, respectively.

Crystallization and preliminary X-ray diffraction analysis of a single variable domain of the immunoglobulin superfamily in amphioxus, Amphi-IgSF-V.

Amphioxus is regarded as an essential animal model for the study of immune evolution. Discovery of new molecules with the immunoglobulin superfamily (IgSF) variable (V) domain in amphioxus would help in studying the evolution of IgSF V molecules in the immune system. A protein was found which just contains only one IgSF V domain in amphioxus, termed Amphi-IgSF-V; it has over 30% sequence identity to the V domains of human immunoglobulins and mammalian T-cell receptors. In order to clarify the three-dimensional structure of this new molecule in amphioxus, Amphi-IgSF-V was expressed, purified and crystallized, and diffraction data were collected to a resolution of 1.95 Å. The crystal belonged to space group P3221, with unit-cell parameters a = b = 53.9, c = 135.5 Å. The Matthews coefficient and solvent content were calculated to be 2.58 Å(3) Da(-1) and 52.38%, respectively. The results will provide structural information to study the evolution of IgSF V molecules in the immune system.

Crystallization and preliminary crystallographic studies of the complement 1qA globular domain from zebrafish, Dare-C1qAgD.

Complement 1q (C1q) is the first component of the complement system which can initiate the classical complement pathway. In human, C1q is composed of 18 polypeptide chains: six C1qA chains, six C1qB chains and six C1qC chains. Each chain has a signal peptide and is comprised of a collagen-like region and a C-terminal C1q globular domain (C1qgD), which is organized as a heterotrimer. C1qgD can recognize antigen-antibody complexes containing IgG and IgM or can bind directly to the C-reactive protein. Although the classical complement pathway is found from fish to mammals, only the human C1qgD structure has been determined. Compared with that of mammals, fish C1q exhibits similar immune functions and genome arrangement. In order to illustrate the structure of C1qgD in fish, zebrafish (Danio rerio) C1qA globular domain (Dare-C1qAgD) was expressed, purified and crystallized. X-ray diffraction data were collected from a crystal to a resolution of 2.05 Å; the crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a=50.347, b=85.059, c=95.560 Å. It contained three molecules in the asymmetric unit. The Matthews coefficient value VM was 2.31 Å3 Da(-1), with a calculated solvent content of 46.7%. The data will help to give insight into the structural basis of C1qA in fish species.

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the bovine CD8αα-BoLA-2*02201 complex.

In order to clarify the structural characteristics of the bovine MHC class I molecule (BoLA-I) complexed with CD8αα (CD8αα-BoLA-I), bovine CD8αα, BoLA-I (BoLA-2*02201) and ß2m were expressed and purified, and were then assembled with a peptide derived from Foot-and-mouth disease virus (FMDV-VP1YY9) and crystallized. The crystal diffracted to 1.7 Šresolution and belonged to space group P21, with unit-cell parameters a=53.9, b=103.8, c=61.8 Å, α=γ=90, ß=96°. The asymmetric unit contained one complex, with a Matthews coefficient of 2.41 Å3 Da(-1) and a solvent content of 48.9%. The rotation-function Z-score and translation-function Z-score for molecular replacement were 3.4 and 8.9, respectively. In addition, SDS-PAGE analysis of CD8αα-BoLA-I crystals showed three bands corresponding to the molecular weights of BoLA-I heavy chain, ß2m and CD8α. The structure of the CD8αα-BoLA-I complex should be helpful in obtaining insight into the interaction between bovine CD8αα and MHC class I molecules. Structure determination of BoLA-2*02201-FMDV-VP1YY9 will be useful in the design of vaccines for foot-and-mouth disease.

Sarcomatoid renal cell carcinoma.

Sarcomatoid renal cell carcinoma (SRCC) is an aggressive tumor variant thought to arise predominantly from differentiation of clear cell carcinoma. A few reports of SRCC associated with non-clear cell tumors led to the presumption that SRCC may arise from any renal cell carcinoma, although direct evidence of this is lacking. We report a case of a 70-year-old male patient, who presented with acute left upper quadrant abdominal pain and was diagnosed to have SRCC after pathological examination. The patient is on high dose interleukin (IL-2)-based immunotherapy and is apparently free of disease six months after surgery.