Appearance of small extracellular vesicles in the mouse pregnant serum and the localization in placentas.

Exosomes or small extracellular vesicles (sEVs) are present in the blood of pregnant mice and considered to be involved in pregnancy physiology. Although sEVs in pregnant periods are proposed to be derived from placentas, sEVs-producing cells are not well known in mouse placentas. We studied the dynamics and localization of sEVs in pregnant serum and placentas, and examined gestational variation of microRNA (miRNA). Serums and placentas were collected from non-pregnant (NP) and pregnant mice throughout the entire gestational day (Gd). EVs were purified from serums and total RNA was isolated from EVs. Nanoparticle-tracking assay (NTA) revealed that the rates of sEVs in EVs are 53% at NP, and increased to 80.1% at Gd 14.5 and 97.5% at Gd 18.5. Western blotting on EVs showed positive reactivity to the tetraspanin markers and clarified that the results using anti-CD63 antibody were most consistent with the sEVs appearance detected by NTA. Serum EVs also showed a positive reaction to the syncytiotrophoblast marker, syncytin-1. Immunohistostaining using anti-CD63 antibody showed positive reactions in mouse placentas at the syncytiotrophoblasts and endothelial cells of the fetal capillaries. Quantitative PCR revealed that significantly higher amounts of miRNAs were included in the sEVs of Gd 18.5. Our results suggested that sEVs are produced in the mouse placenta and transferred to maternal or fetal bloodstreams. sEVs are expected to have a miRNA-mediated physiological effect and become useful biomarkers reflecting the pregnancy status.

Specific expression and blood kinetics for relaxin 2, lipocalin 2, and tissue factor pathway inhibitor 2 at the canine placenta and pregnant bloods.

In general, humoral factors released from the placenta influence pregnancy progression, but the involvement of the canine placenta is often unidentified. We investigated specific genes in canine placentas and analyzed the blood dynamics of the translated proteins. Furthermore, RNAs are known to be released from placentas embedding in exosomes, a type of extracellular vesicles. Here, the presence of cell-free RNAs in pregnant serums was also confirmed. RNA specimens were purified from the normal healthy dog placentas and applied to RNA-Seq analysis. Expressions of frequent genes were confirmed by RT-PCR using placentas from other individuals and breeds. Relaxin (RLN) 2, lipocalin (LCN) 2, and tissue factor pathway inhibitor (TFPI) 2 were selected as high-expressed and placenta-specific genes. By western blot, the three factors were clearly detected in the pregnant serums. Quantitative analysis revealed that the amount of RLN2 increased significantly from non-pregnancy to day 41 of pregnancy. Regarding LCN2 and TFPI2, the protein serum levels elevated during pregnancy, but the statistical differences were not detected. Exosomes were found in all pregnant serums; however, the percentage was less than 6% in total extracellular vesicles. The cell-free RNA related to RLN2 was detected, but no elevation was confirmed during pregnancy. We found specific genes in the canine placenta and the transition of their translated protein into the blood. These factors may become useful tools for research on canine pregnancy and monitoring of reproductive management. Exosomes and cell-free RNA could not be found to be valid in canine reproduction.

Molecular identification of blaTEM and blaCTX-M genes in multidrug-resistant Escherichia coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia.

Introduction: Escherichia coli is an opportunistic bacteria that can grow easily, produce toxins, and resist antibiotics. The phenomenon of E. coli developing multidrug resistance is currently the subject of extensive research. The objective of this study was to molecularly identify blaTEM and blaCTX-M genes in multidrug-resistant E. coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia. Material and Methods: One hundred and ten milk samples were collected from 45 dairy cattle farms in Tulungagung, Indonesia. Indole, methyl red, Voges-Proskauer and in citrate tests and triple iron sugar agar tests were used to identify E. coli. Multidrug resistance was determined in isolates through antibiotic sensitivity tests using tetracycline, streptomycin, trimethoprim, chloramphenicol and aztreonam. Extended-spectrum beta lactamase enzyme production was confirmed by double-disc synergy test (DDST). Molecular identification was performed to confirm the blaTEM and blaCTX-M genes. Results: One hundred and one (91.82%) E. coli strains were isolated from the samples. The antibiotic sensitivity test showed four (3.96%) multidrug-resistant (MDR) and one (0.99%) ESBL-positive E. coli by DDST confirmation. There were three (77.78%) blaTEM genes and one (0.99%) blaCTX-M gene discovered in the MDR E. coli isolates using PCR for molecular identification. Conclusion: The findings of the blaTEM and blaCTX-M genes encoding ESBL E. coli in dairy cattle milk in Tulungagung, Indonesia is concerning and argues for prompt action to stop the emergence of antibiotic resistance which has an impact on public health.

Expression dynamics of Crry at the implantation sites in normal pregnancy and response against miscarriage induction.

In mammals, immune tolerance against fetal tissue has been established for normal pregnancy progression. It is known that Crry regulates complemental activity to prevent injury of the mouse embryo and extra-embryonic tissue. This study aimed to examine the expression appearance and normal localization sites of Crry in the mouse placenta. Also, the emergency responses of Crry were verified at the time of experimental miscarriage induction. Moreover, we investigated an existing similar protein of Crry in animal placentas other than mice. Crry expression level showed a peak at day 8.5 of pregnancy. Trophoblast giant cells and decidual cells indicated a positive reaction to anti-Crry antibody. After treatments of interferon-γ, Crry expression was increased significantly in the survived implantation sites as compared with the controls. However, there was no significant difference in the miscarriage-initiated sites. It disclosed that Crry distributes from the early to middle periods of the placentas and involves complement regulation at the extraembryonic tissue and decidua basalis. Crry also showed an ability to respond to risk against external initiation for urgent miscarriage. Finally, we found anti-mouse Crry antibody-bound proteins in the placenta of many animals. It suggests a potency of Crry to make an environment of immune tolerance in many types of mammal placentas.

Morphology and histology of paryphasmata and hemibaculum of Varanus salvator based on sexual maturity.

Background: Varanus salvator is one of the reptiles being hunted by human beings for several purposes, including traditional medicine. The studies about reproductive biology aspects were limited. Aim: This study aimed to determine the morphology, histology, and histometry of V. salvator paryphasmata and hemibaculum based on Snout-Vent Length (SVL) as an indicator of sexual maturity. Methods: This study examined 18 pairs of hemipenis of V. salvator with SVL more and less than 40 cm in equal number. Paryphasmata and hemibaculum parts were observed visually and micro-sliced, then stained with Hematoxylin-Eosin (HE). The histological observation was conducted under a 40×, 100×, and 400× magnification of a light microscope. The histometry of the paryphasmata was examined using 13 Megapixels Coolpad and OptiLab Plus for microscopic pictures. The chondrocyte cell area was measured using the Optilab Plus and Image Raster three applications. Results: The sizes of glans of hemipenis, paryphasmata, and hemibaculum increased according to the increasing of SVL. The average paryphasmata row number, epidermis, and loose connective tissue thickness were not significantly different (p > 0.05). However, dense connective tissue was thicker (p < 0.05), which corresponds to SVL. Hemibaculum was composed of fibrous and hyaline cartilage characterized by chondrocyte cells. The SVL also affects (p < 0.05) the ossification of hyaline in hemipenis, while the chondrocyte cell area followed the equation -1.87E7 + 7.09E5* SVL. Conclusion: The SVL size of V. salvator affects the paryphasmata, hemibaculum, thickness of dense connective tissue of paryphasmata, and the area of chondrocyte cells.

First report of three multivalvulid species (Cnidaria: Myxozoa: Myxosporea) in commercial fishes from Java Sea, Indonesia, with records of Unicapsula pyramidata and two new Kudoa spp.

Commercial marine fishes caught locally in East Java, Indonesia, were examined for multivalvulid myxosporeans (Cnidaria: Myxozoa: Myxosporea). Plasmodia of Unicapsula pyramidata were detected in the trunk muscle of two fork-tailed threadfin breams (Nemipterus furcosus). Genetic comparisons of this sample to those collected in the Australian Coral Sea and South China Sea showed few nucleotide substitutions in the small subunit and large subunit ribosomal RNA gene (rDNA) with the species isolated in the Australian Coral Sea and South China Sea. Pseudocysts of two new Kudoa spp. with four shell valves and polar capsules were found in the trunk muscle of two shrimp scads Alepes djedaba and two flathead grey mullets Mugil cephalus. Kudoa javaensis n. sp. myxospores isolated from the shrimp scad were 5.1-7.2 (mean 6.2) µm thick, 6.2-7.9 (7.3) µm wide, and 4.6-6.3 (5.4) µm long, with polar capsules 1.9-2.5 (2.2) µm long and 1.1-1.4 (1.3) µm wide (n = 15). Kudoa surabayaensis n. sp. myxospores isolated from the flathead grey mullet were 5.8-6.7 (6.3) µm thick, 6.4-7.6 (6.9) µm wide, and 4.6-5.0 (4.7) µm long, with polar capsules 1.8-2.4 (2.1) µm long and 0.9-1.3 (1.1) µm wide (n = 25). These two Kudoa spp. showed critical differences in spore shapes (semiquadrate with unequal shell valves vs. equal shell valves), and absence vs. presence of uplifted shell valve termini. Nucleotide sequencing of rDNA supported the morphological differentiation of these two species. Furthermore, these two isolates were morphologically and phylogenetically distinct from any recorded Kudoa spp.

Phenotypic approach artemisinin resistance in malaria rodent as in vivo model.

AIM: The aim of this study is to prove the development of artemisinin resistance phenotypically in malaria rodent as an in vivo resistance development model in humans. MATERIALS AND METHODS: Plasmodium berghei was infected intraperitoneally in mice, then artemisinin was given with "4-day-test" with effective dose (ED) 99% dose for 3 days which begins 48 h after infection (D2, D3, and D4). Parasite development was followed during 5th until 10th days of infection. After parasitemia >2% of red blood cell which contains parasites on 1 mice, that mice were used as donor to be passaged on the new 5 mice. After that, parasitemia was calculated. ED50 and ED90 were examined with parasite clearance time (PCT), recrudescence time (RT), and also morphology development examination of intraerythrocytic cycle of P. berghei with transmission electron microscope. RESULTS: Among the control group compare with the treatment group showed significant differences at α=0.05 on 5th day (D5) until 10th day (D10). The control group of 4th passage (K4) with passage treatment group of 4th passage (P4) on the 10th days (D10) post infection showed no significant differences in the α=0.05. The average percentage of inhibition growth was decreasing which is started from 5th to 10th day post infection in P1, P2, P3, and P4. On the development of P. berghei stage, which is given repeated artemisinin and repeated passage, there was a formation of dormant and also vacuoles in Plasmodium that exposed to the drug. CONCLUSION: Exposure to artemisinin with repeated passages in mice increased the value of ED50 and ED90, decreased the PCT and RT and also changes in morphology dormant and vacuole formation.