BACKGROUND: Reports indicate that H.pylori is able to invade the eukaryotic cells and establish inside their vacuoles. In this study, FITC-conjugated IgY-Hp was used to localize H.pylori inside the vacuole of Candida yeast. Presence of intracellular H.pylori inside the new generations of yeast cells was also examined by light microscopy and Live/Dead BacLight staining method.METHODS: A single colony of fresh yeast culture was cultivated in a 100-ml medium containing yeast extract and N-acetylglucoseamine supplemented with fetal bovine serum. After 12-hr incubation at 37oC, FITC-conjugated IgY-Hp was added. After 3 hours, 10 mL of yeast suspension was smeared on a glass slide, air-dried and examined by fluorescent microscopy. Wet mounts of yeast culture and Live/Dead BacLight stained preparations were examined by light and fluorescent microscopy, respectively. Photographs were taken from the fast-moving H.pylori inside the yeast vacuoles.RESULTS: Fluorescent microscopy showed that FITC-conjugated IgY-Hp could enter yeast cells and specifically react with H.pylori, localizing the bacterium inside the yeast vacuole. Photographs taken from wet mounts observed by light and fluorescent microscopy showed fast-moving H.pylori cells in the vacuole of mother as well as daughter yeast cells. The intravacuolar H.pylori cells stained green, showing their viability.CONCLUSION: Intracellular life of prokaryotes inside eukaryotes has been described as an evolutionary phenomenon with a great impact on bacterial persistence despite environmental stresses. Results of this study demonstrated the specific interaction of FITC-conjugated IgY-Hp with H.pylori cells and the bacterial localization inside the Candida yeast vacuole. The intracellular bacteria were viable and existed in the vacuole of next generations of yeast cells. It appears that H.pylori is well-equipped to dwell within the vacuole of eukaryotic cells where it is protected from stressful conditions, including antibacterial therapy. Presence of H.pylori inside the vacuole of new generations of yeasts demonstrates the intimate relationship between the two microorganisms, resulting in bacterial inheritance as part of the vacuolar content of yeast cells.

Purpose: To introduce a specular microscopic reference image for endothelial vacuolation in donated corneas. Methods: Two corneas from a donor with diffuse, round to oval dark areas at the endothelial level on slit lamp biomicroscopy and one normal-appearing donor cornea underwent specular microscopy, histopathologic evaluation and transmission electron microscopy. Results: Specular microscopy of the two corneas with abnormal-looking endothelium revealed large numbers of dark, round to oval structures within the endothelium in favor of endothelial vacuolation. Light microscopy disclosed variable sized cyst-like structures within the cytoplasm. Transmission electron microscopy showed electronlucent and relatively large-sized intracytoplasmic vacuoles. These features were not observed in the endothelium of the normal cornea. Conclusion: The specular microscopic features of endothelial vacuolation in donated corneas were confirmed by light microscopy and transmission electron microscopy, therefore the specular image may be proposed as a reference to eye banks.

Objective: It is necessary to evaluate fertility effective agents to predict assisted reproduction outcomes. This study was designed to examine sperm vacuole characteristics, and its association with sperm chromatin status and protamine-1 (PRM1) to protamine-2 (PRM2) ratio, to predict assisted pregnancy outcomes. Materials and Methods: In this experimental study, ninety eight semen samples from infertile men were classified based on Vanderzwalmen’ s criteria as follows: grade I: no vacuoles; grade II: ≤ 2 small vacuoles; grade III: ≥ 1 large vacuole and grade IV: large vacuole with other abnormalities. The location, frequency and size of vacuoles were assessed using high magnification, a deep learning algorithm, and scanning electron microscopy (SEM). The chromatin integrity, condensation, viability and acrosome integrity, and protamination status were evaluated for vacuolated samples by toluidine blue (TB) staining, aniline blue, triple staining, and CMA3 staining, respectively. Also, Protamine-1 and protamine-2 genes expression was analysed by reverse transcription-quantitative polymerase chain reaction (PCR). The assisted reproduction outcomes were also followed for each cycle. Results: The results show a significant correlation between the vacuole size (III and IV) and abnormal sperm chromatin condensation (P=0. 03 and P=0. 02, respectively), and also, protamine-deficient (P=0. 04 and P=0. 03, respectively). The percentage of reacting acrosomes was significantly higher in the grades III and IV spermatozoa in comparison with normal group. The vacuolated spermatozoa with grade IV showed a high protamine mRNA ratio (PRM-2 was underexpressed, P=0. 01). In the IVF cycles, we observed a negative association between sperm head vacuole and fertilization rate (P=0. 01). This negative association was also significantly observed in pregnancy and live birth rate in the groups with grade III and IV (P=0. 04 and P=0. 03, respectively). Conclusion: The results of our study highlight the importance sperm parameters such as sperm head vacuole characteristics, particularly those parameters with the potency of reflecting protamine-deficiency and in vitro fertilization/ intracytoplasmic sperm injection (IVF/ICSI) outcomes predicting.

BACKGROUND: Human gastric epithelium and immunocytes have been recognized as the sole specialized eukaryotic cells that host Helicobacter pylori (H. pylori). The aim of this study was to provide further evidence for our previous proposal regarding the occurrence of H.pylori inside the yeast vacuole, verifying the viability of the intravacuolar H.pylori by western blotting.METHODS: Light microscopy and polymerase chain reaction (PCR) were used for primary detection of nonculturable H.pylori in 11 Candida yeasts (six oral and five gastric). Boiling was used for extraction of proteins from yeasts and the control H.pylori. Western blot analysis was recruited to assess the occurrence of H.pylori-specific proteins in protein pool of yeasts, using IgY-Hp raised in hens and IgG1-Hp raised in mice.RESULTS: The fast-moving bacterium-like bodies (BLBs) were identified as H.pylori by amplification of H.pylori 16S rRNA, ureAB, vacA s1, and ahpC genes from the whole DNA of yeasts. Analysis of the sequenced products of 16S rRNA gene amplified from the yeast and H.pylori isolates of patient #2 showed 100% homology with the corresponding sequences of the reference H. pylori strains in GenBank. According to published data, it was plausible to assign the H.pylori-specific proteins, detected by western blot analysis, as thiol peroxidase (21 kDa), peroxiredoxin (AhpC) (26 kDa), urease-A subunit (UreA) (32 kDa), vacuolating cytotoxin A (VacA) small subunit (36 kDa), and VacA large subunit (56 kDa). CONCLUSION: Results of this study show that inside yeast, H.pylori expresses proteins and is viable. These proteins appear to serve as powerful tools to help H.pylori to establish in the vacuole of yeast where it can reach nutrients and multiply. The intimate relationship between H.pylori and Candida yeast which began long time ago, could have led to the establishment of H.pylori inside the yeast vacuole before invading human cells.

Although intracytoplasmic sperm injection (ICSI) allows proper fertilization in most cases of male sub fertility, it is one of the most unphysiological techniques in assisted reproductive technologies (ART). Thus, over the last decade, researchers have tried to improve sperm observation with higher-resolution microscopy techniques such as the intracytoplasmic morphologically selected sperm injection (IMSI) technique. In order to identify literatures for this review, the PubMed database was searched from 2000 onwards using the terms IMSI, motile sperm organelle morphology examination (MSOME) and sperm vacuole. Approximately 10 years after the introduction of the MSOME and IMSI procedures, several questions related to the prevalence, origin, location, and clinical consequences of sperm vacuoles have not yet been clarified. It seems that IMSI as a routine application is not state of the art and the only confirmed indications for IMSI are recurrent implantation failure following ICSI and severe male factor.