Cytoplasmic Extrusions Research Articles

The Clinical Management of Leukocytospermia in Male Infertility: A Narrative Review

A major global health concern, male infertility affects 8–12% of couples globally. Leukocytospermia is a complicated illness that is distinguished from other reasons causing male infertility by having high white blood cell counts in semen. The complex mechanisms behind leukocytospermia’s effects on sperm function and fertility are examined in this review. Leukocytospermia induces oxidative stress and reactive oxygen species (ROS) that impair DNA integrity, mitochondrial function, cytoplasmic extrusion, and sperm quality overall. Leukocytospermia is exacerbated by non-infectious factors, such as substance abuse and varicocele, even though genital tract infections are a common cause. The usefulness and dependability of diagnostic techniques range from immunochemistry to direct counting. Although there is still disagreement on the most effective course of action, clinical-care techniques, such as antioxidant supplementation and antibiotic therapy, attempt to address underlying causes and reduce ROS-induced damage. Prospectively, the combination of artificial intelligence with the latest developments in artificial reproductive technologies presents opportunities for more precise diagnosis and customized treatments.

Effects of Different Types of Recombinant SARS-CoV-2 Spike Protein on Circulating Monocytes' Structure.

This study investigated the biological effects on circulating monocytes after challenge with SARS-CoV-2 recombinant spike protein. Whole blood collected from seven ostensibly healthy healthcare workers was incubated for 15 min with 2 and 20 ng/mL final concentration of recombinant spike protein of Ancestral, Alpha, Delta, and Omicron variants. Samples were analyzed with Sysmex XN and DI-60 analyzers. Cellular complexity (i.e., the presence of granules, vacuoles and other cytoplasmic inclusions) increased in all samples challenged with the recombinant spike protein of the Ancestral, Alpha, and Delta variants, but not in those containing Omicron. The cellular content of nucleic acids was constantly decreased in most samples, achieving statistical significance in those containing 20 ng/mL of Alpha and Delta recombinant spike proteins. The heterogeneity of monocyte volumes significantly increased in all samples, achieving statistical significance in those containing 20 ng/mL of recombinant spike protein of the Ancestral, Alpha and Delta variants. The monocyte morphological abnormalities after spike protein challenge included dysmorphia, granulation, intense vacuolization, platelet phagocytosis, development of aberrant nuclei, and cytoplasmic extrusions. The SARS-CoV-2 spike protein triggers important monocyte morphological abnormalities, more evident in cells challenged with recombinant spike protein of the more clinically severe Alpha and Delta variants.

Fungicolous Fungi on Pseudosclerotial Plates and Apothecia of Hymenoscyphus fraxineus and Their Biocontrol Potential.

In the present work, research tasks were carried out in the search for fungi with potential biocontrol possibilities in relation to the ash dieback pathogen, Hymenoscyphus fraxineus. In the years 2012-2021, dead petioles of F. excelsior and F. mandshurica were collected, on which morphological structures of H. fraxineus showed unusual symptoms of dying (apothecia) and signs of colonization by other fungi (pseudosclerotial plates). Based on morphological and molecular phylogenetic data, 18 fungal taxa were identified. Thirteen of them belong to Ascomycota: Clonostachys rosea, Cl. solani, Cordyceps sp., Minimidochium sp., Nemania diffusa, Fusarium sp., Pestalotiopsis sp., Trichoderma atroviride, T. harzianum, T. polysporum, T. rodmanii, T. tomentosum, Trichoderma sp., and five other taxa are represented by Basidiomycota: Corticiales sp., Cyathus olla, Efibula sp., Gymnopus sp. and Polyporales sp. In 108 dual cultures in vitro, three different types of interactions were distinguished: (i) physical colony contact (5.6%), (ii) presence of an inhibition zone between the colonies (0.9%), and (iii) copartner overgrowth of H. fraxineus colonies and partial or complete replacement of the pathogen (93.5%). In the dual cultures, various morphological deformations of H. fraxineus hyphae were observed: the development of apical or intercalary cytoplasmic extrusions, development of internal hyphae of the test fungi in pathogens' hyphae, the deformation and disruption of significant sections of H. fraxineus hyphae via lysis and mycoparasitism, complete desolation of H. fraxineus cells and breakdown of hyphae into short fragments, and disappearing of pigment in the affected hyphae of H. fraxineus. The inoculation tests performed in vivo or in glass Petrie dishes showed that all the identified taxa were able to lead to pathological changes in H. fraxineus apothecia, and the mycelium of some of them completely covered pseudosclerotial plates of H. fraxineus. It was emphasized in the discussion that such activity of these fungi in forest stands may contribute to the reduction in the H. fraxineus inoculum reservoir.

Fungal endophytes in Fraxinus excelsior petioles and their in vitro antagonistic potential against the ash dieback pathogen Hymenoscyphus fraxineus.

Fungal endophytes were isolated from 250 asymptomatic leaf petioles of Fraxinus excelsior collected from trees showing symptoms of ash dieback in five forest sites in southern Poland. Fungal isolations yielded 1646 colonies representing 97 taxa, including 92 Ascomycota and 5 Basidiomycota species. The most common Ascomycota comprised Nemania serpens (38.0 % of colonized petioles), Diaporthe eres (33.6 %), Venturia fraxini (26.4 %), Diaporthe sp. 1 (20.4 %), Alternaria sp. 1 (14.8 %), Colletotrichum acutatum (14.8 %), Nemania diffusa (14.0 %), Colletotrichum gloeosporioides (12.4 %) and Colletotrichum sp. (12.4 %). The occurrence of all these taxa except Alternaria sp. 1 was significantly different between the studied forest sites. Two yeast species, Vishniacozyma foliicola (4.8 %) and Cystobasidium pinicola (2.8 %), dominated among the Basidiomycota endophytes detected. All the fungal endophytes were tested in dual culture antagonistic assays against two strains of Hymenoscyphus fraxineus, resulting in the development of four interaction types. The interactions included the physical contact of co-partners' mycelia (41.8 %), development of an inhibition zone (47.4 %), growth of endophyte mycelia over H. fraxineus colonies (9.3 %) and growth of H. fraxineus mycelia over endophyte colonies (1.5 %). The strongest antibiotic activity against H. fraxineus, measured by the width of the inhibition zone, was observed for Cytospora pruinosa, Fusarium lateritium, Phoma sp. 2, Pleosporales sp. 2 and Thielavia basicola. A variety of morphophysiological deformations of H. fraxineus hyphae were observed under endophyte pressure: spiral twist of the hyphae, formation of cytoplasmic extrusions, development of torulose hyphae and excessive lateral branching of the hyphae. The strongest antagonistic effects, coupled with the potential to overgrow H. fraxineus colonies, was shown by Clonostachys rosea, Nemania diffusa, N. serpens, Peniophora cinerea, Rosellinia corticium and Xylaria polymorpha. Some of these species were able to attack H. fraxineus hyphae in a mycoparasitic manner. The antagonistic activities included the physical penetration of H. fraxineus hyphae, dissolution of hyphal cell walls, disappearance of pigmentation, disintegration of hyphae and degradation of other fungal structures. In contrast, one of the most commonly detected endophytes in ash leaves, Venturia fraxini, did not show in vitro antagonistic potential against H. fraxineus. Finally, we discuss the potential of the detected fungal endophytes to combat H. fraxineus invasion, the cause of ash decline in Europe.

P–142 Comparison of the IVF outcome in the course of ICSI vs PICSI treatment continuation in exactly the same group of HBA abnormal patients

Abstract Study question Does the PICSI have a beneficial effect for men with abnormal HBA on the fertilization rate, blastocysts number and clinical pregnancies in the next attempt? Summary answer Patients with HBA <80% choosing to undergo PICSI after ICSI failure see an increase in blastocyst and pregnancy rates. What is known already Hyaluronic acid (HA) is a main component of cervical mucus and the extracellular matrix of cumulus cells. The formation of HA-binding sites in sperm cell membranes is one of the markers of sperm maturation indicating completion the spermatogenic process of remodelling the plasmatic membrane, cytoplasmic extrusion and nuclear maturity. Spermatozoa selected by the HA-binding technique (the physiologically selected intracytoplasmic sperm injection – PICSI) have a potentially reduced risk of chromosomal aneuploidy or DNA fragmentation. Recent evidence do not show significant benefits in using PICSI. However, it has not been analysed in the course of treatment continuation in the same patients. Study design, size, duration This was a retrospective case-control study. It included exactly the same 58 patients with abnormal HBA, who underwent IVF treatment with ICSI initially and later with PICSI, between January 2014 and October 2020 at INVICTA Fertility Centre, Poland. Median female partner age in PICSI group was 36,2±5,34, without PICSI 35,8 ±5,28. Participants/materials, setting, methods 275 cycles (130 ICSI and 145 PICSI) resulted in 793 and 897 MII respectively. Patients were also divided into two groups <80% and ≥80% depending on the obtained HBA score expressed as the percentage of sperm bound with hyaluronan. The analysis covered the fertilization rate (FR), TQ and total blastocyst rate on day 5 and clinical pregnancy rate. Patients with poor response to stimulation were excluded from the study. Main results and the role of chance FR in ICSI and PICSI groups was not significantly difference (57.00%±31.2 vs 59.87%±30.8) even when taking into account the division of patients according to the obtained HBA score. In the <80% group the FR was 57.04%±29.3 vs 59.54%±30.8 in ICSI vs PICSI group respectively. There were no significant differences when comparing the under HBA ≥80% subgroups for all analysed outcomes. Fertilization rate ​​was 56.88% in the ICSI group vs 61.03% in the PICSI group. The percentage of blastocysts was 28.61% vs 34.45% and the percentage of TQ blastocysts on day 5 was 15.32% vs 16.81% with ICSI and PICSI respectively, in the group consisting of the same patients. In the HBA <80% group significant differences were observed in the percentage of obtained blastocysts 37.81% vs 47.61% by comparing the ICSI and PICSI approaches (p < 0.05). Also, percentage of TQ blastocyst on day 5 also was higher in patients with <80% HBA score after PICSI and was statistically significant (17.07% ICSI vs 23.92% PICSI, p < 0.05). We saw statistically significant (p < 0.01) increase in percentage of clinical pregnancies from 29.03% without PICSI to 69.44% in patient’s subsequent procedures involving PICSI. Limitations, reasons for caution More data is required to confirm that improved results of PICSI procedure are consistent and possible to reproduce in a larger group – and as a result could be included as part of the standard treatment process. Wider implications of the findings: The presented results show that in patients with normal HBA score, PICSI does not bring a measurable benefit and this may be important factor to consider in decision-making for couples seeking assistance. Trial registration number Not applicable

Functional Validation of DownRegulated MicroRNAs in HeLa Cells Treated with Polyalthia longifolia Leaf Extract Using Different Microscopic Approaches: A Morphological Alteration-Based Validation.

Several microscopy methods have been developed to assess the morphological changes in cells in the investigations of the mode of cell death in response to a stimulus. Our recent finding on the treatment of the IC50 concentration (26.67 μg/mL) of Polyalthia longifolia leaf extract indicated the induction of apoptotic cell death via the regulation of miRNA in HeLa cells. Hence, the current study was conducted to validate the function of these downregulated microRNAs in P. longifolia-treated HeLa cells using microscopic approaches. These include scanning electron microscope (SEM), transmission electron microscope (TEM), and acridine orange/propidium iodide (AO/PI)-based fluorescent microscopy techniques by observing the morphological alterations to cells after transfection with mimic miRNA. Interestingly, the morphological changes observed in this study demonstrated the apoptotic hallmarks, for instance, cell blebbing, cell shrinkage, cytoplasmic and nuclear condensation, vacuolization, cytoplasmic extrusion, and the formation of apoptotic bodies, which proved the role of dysregulated miRNAs in apoptotic HeLa cell death after treatment with the P. longifolia leaf extract. Conclusively, the current study proved the crucial role of downregulated miR-484 and miR-221-5p in the induction of apoptotic cell death in P. longifolia-treated HeLa cells using three approaches-SEM, TEM, and AO/PI-based fluorescent microscope.

Epididymal Toxicity Associated with Vincristine Treatment

Vincristine, a major player in front line combination chemotherapy of cancer reduces testosterone levels contributing to reproductive toxicity. Much is known about testicular toxicity of vincristine as compared to its effect on epididymis; hence, the present study aimed to evaluate the epididymal toxicity associated with vincristine treatment, which also contributes to the overall reproductive toxicity associated with vincristine. Vincristine was intraperitoneally injected to adult male Wistar rats of proven fertility with a dose of 40 mg/kg/day dissolved in 0.5 ml of physiological saline for 30 days. The epididymal weight was found to be unaltered after treatment whereas sperm count was reduced significantly. Significant changes were noted in ion concentrations of cauda and caput of epididymis with changes in protein profile of the tissue, sperm and luminal protein from cauda and caput region, which plays a significant role in sperm maturation and sperm transport. Infertility associated with vincristine could be attributed to its effects on various epididymal proteins involved in sperm protection and various stages of sperm development such as cytoplasmic extrusion and membrane stabilization, which had contributed to the abnormal sperm count and impaired function.

Isolation and characterization of halotolerant bacilli from chickpea (Cicer arietinum L.) rhizosphere for plant growth promotion and biocontrol traits

The present study describes the identification and polyphasic characterization of salt tolerant bacilli displaying plant growth promoting and broad spectrum antifungal activities. A total of 110 strains were isolated, out of which nine were selected on the basis of halotolerance, in vitro plant growth promoting attributes and antagonism against multiple phytopathogens. Preliminary identification of strains was done on the basis of phylogenetic analysis and comparison of 16S RNA sequences with type strains. The test strains were identified as Bacillus pumilus, B. subtilis, B. licheniformis, B. safensis and B. cereus. These strains possess ability to tolerate high salt (8–10% NaCl), form endospore, showed broad spectrum antifungal activity and therefore might cope with adverse environments. Scanning electron microscopy of promising test strains (CG18 and CM25) and pathogen interaction indicated destruction of fungal mycelia by halotolerant antagonists due to cytoplasmic extrusion. Moreover, PCR amplification of bacillomycin (bmyB), chitinase (ChiA), and β-glucanase genes and production of hydrolytic enzymes suggested that test strains could have a role to manage plant pathogens under saline conditions. Finally, the selected strains exhibited some plant growth promotion traits. Overall, this study demonstrated that the evaluated strains could be useful in developing microbial products to enhance tolerance to biotic and abiotic stresses and boost plant growth.

Histological and immunohistochemical study of the role of stem cells, conditioned medium and microvesicles in treatment of experimentally induced acute kidney injury in rats

Background: Acute kidney injury (AKI) is a major health problem associated with high morbidity and mortality rates.Mesenchymal stem cells (MSCs) have revealed advantages for therapeutic use in medical practice. Microvesicles (MVs)are membranous, cell-derived vesicles released by MSCs into their microenvironment. The conditioned medium (CM) isthe medium surrounding MSCs. Aim of the Work: This study aimed to investigate the therapeutic potential of MSCs, their CM and microvesicles (MVs) on experimentally induced acute kidney injury in rats. Materials and Methods: Fifty-five rats were divided into five groups: Group I (control group). Group II: given glycerol intramuscularly. Group III: given glycerol then MSCs. Group IV: given glycerol then CM. Group V: given glycerol then MVs. Kidney specimens were processed for H&E and Ki-67 staining and EM studies. Results: Subgroup IIA revealed vacuolation of the cytoplasm, flattening of the epithelial lining the tubules, extrusion of cytoplasm and nuclei into luminal spaces, deeply stained nuclei, and hyaline material in tubular lumina. EM examination of proximal and distal convoluted tubules showed multiple vacuoles, lysosomes, loss of continuity of apical cell membrane,presence of debris in the lumina and vacuolated mitochondria. Group IIB revealed poor improvement with persistence of most lesions. Groups III, VI and V showed amelioration of most of these lesions, and decrease in blood urea and serum creatinine levels. Conclusion: Mesenchymal stem cells, CM and MVs ameliorate induced AKI and with little differences in their effectiveness. CM and MVs can be used in treating diseases.

Standardized Polyalthia longifolia leaf extract (PLME) inhibits cell proliferation and promotes apoptosis: The anti-cancer study with various microscopy methods.

Over the years a number of microscopy methods have been developed to assess the changes in cells. Some non-invasive techniques such as holographic digital microscopy (HDM), which although does not destroy the cells, but helps to monitor the events that leads to initiation of apoptotic cell death. In this study, the apoptogenic property and the cytotoxic effect of P. longifolia leaf methanolic extract (PLME) against the human cervical carcinoma cells (HeLa) was studied using light microscope (LM), holographic digital microscopy (HDM), scanning electron microscope (SEM) and transmission electron microscope (TEM). The average IC50 value of PLME against HeLa cells obtained by MTT and CyQuant assay was 22.00μg/mL at 24h. However, noncancerous Vero cells tested with PLME exhibited no cytotoxicity with the IC50 value of 51.07μg/mL at 24h by using MTT assay. Cytological observations showed nuclear condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, disappearance of microvilli and filopodia, narrowing of lamellipodia, holes, formation of numerous smaller vacuoles, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by HDM, LM, SEM and TEM. In conclusion, PLME was able to produce distinctive morphological features of HeLa cell death that corresponds to apoptosis.

225 - Expansion of Bone Marrow NK Lymphocytes is Associated with Higher Probability of Response to Azacitidine Treatment and Survival Benefit

Over the years a number of microscopy methods have been developed to assess the changes in cells. Some non-invasive techniques such as holographic digital microscopy (HDM), which although does not destroy the cells, but helps to monitor the events that leads to initiation of apoptotic cell death. In this study, the apoptogenic property and the cytotoxic effect of P. longifolia leaf methanolic extract (PLME) against the human cervical carcinoma cells (HeLa) was studied using light microscope (LM), holographic digital microscopy (HDM), scanning electron microscope (SEM) and transmission electron microscope (TEM). The average IC50 value of PLME against HeLa cells obtained by MTT and CyQuant assay was 22.00 μg/mL at 24 h. However, noncancerous Vero cells tested with PLME exhibited no cytotoxicity with the IC50 value of 51.07 μg/mL at 24 h by using MTT assay. Cytological observations showed nuclear condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, disappearance of microvilli and filopodia, narrowing of lamellipodia, holes, formation of numerous smaller vacuoles, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by HDM, LM, SEM and TEM. In conclusion, PLME was able to produce distinctive morphological features of HeLa cell death that corresponds to apoptosis.

Embryonic development of the saccular sensory epithelium in relation to otolith growth in the inner ear of the silver carp, Hypophthalmichthys molitrix (Valenciennes, 1844) (Teleostei: Cyprinidae): light and electron microscopic study

The development of the saccular sensory epithelium in the inner ear of the silver carp, Hypophthalmichthys molitrix, was studied from an early stage (2 days after fertilization), when the otic vesicle or otocyst first formed, to the post-larval stage (7 days after hatching), when the development of the inner ear approximates that of the juvenile stage. Light microscopy revealed: (1) the otic vesicle, the primordia of the saccular otolith and the stato-acoustic ganglion were observed by 2 days after fertilization; (2) the saccular macula overlain by a small round otolith is established at 3 days after fertilization, while the utricular macula with its otolith is first observed at 4 days after fertilization; (3) the saccular macula began to differentiate at 3 days after hatching and became well differentiated at 7 days after hatching. At 7 days after hatching, transmission electron microscopy (TEM) revealed: (1) the apical surface of each hair cell is covered with a ciliary bundle formed of a long kinocilium and a short bundle of stereocilia. The supporting cells are provided with microridges on their apical surface and seemed to be covered with small and large vesicles, suggesting that they have a secretory function beside the supporting one; (2) secretory materials such as multivesicular bodies, electron dense granules, empty vesicles and cytoplasmic extrusions are observed scattering over the saccular sensory epithelium, which probably contribute to the formation of the otolith and/or otolithic membrane. Using scanning electron microscopy (SEM), two types of hair bundles are distinguished: the first type consisted of numerous short stereocilia and a kinocilium; the second type has a small number of short stereocilia with a very long kinocilium. Most of these results are discussed with special regard to the environmental factors affecting the early development of the inner ear in teleost fishes.

Cloned Sheep Blastocysts Derived from Oocytes Enucleated Manually Using a Pulled Pasteur Pipette

The potential applications of a simplified method of somatic cell nuclear transfer (SCNT) that is improved in both efficiency and throughput is considerable. Technically, a major step of SCNT is to produce large pools of enucleated oocytes (cytoplasts) efficiently, a process that requires considerable micromanipulation skill and expensive equipment. Here, we have developed an efficient and high-throughput method of manual oocyte enucleation using a simple device, a pulled Pasteur pipette, that can be connected to standard zona-free method of embryo reconstitution. Common Pasteur pipettes were pulled on a flame to produce finely drawn pipettes with inner diameters approximately less than half the oocyte diameter (∼50-60 μm), and slightly larger than cytoplasmic protrusion (∼20-30 μm) that was induced after demecolcine treatment of MII-stage oocytes. Oocyte manipulation was performed under a stereomicroscope by either bisecting the oocyte into two approximately equal demioocytes (blind manual enucleation), or by positioning the oocytes so that the cytoplasmic extrusion that contains the MII chromosome mass is removed with the minimum amount of cytoplasm (oriented manual enucleation). The survival rate of the manually enucleated oocytes was 81.4-91.5%, comparable to standard zona-free method of oocyte enucleation (>95%). A total of 80-120 oocytes could be enucleated in 10 min, which was considerably higher than standard zona-free enucleation method. In vitro development rates of cloned embryos derived from manually enucleated cytoplasts with varying cytoplasmic volumes (50%, 95%, and 100%) was comparable, and embryonic developmental rates of the two latter groups were at least as good as standard zona-free method. The manual method of oocyte enucleation described here can be learned and mastered for simple, fast, and cheap production of cloned embryos with comparable efficiency to other available methods.

Zona Pellucida Ablation for Day 3 Embryo Hatching in Poor Prognosis Cases Prior to Acellular Fragment Removal: Acidified Tyrodes Vs. Non-Contact 500μS Laser Pulsation

Assisted hatching (AH) of human embryos has commonly been performed using the acidified media technique (AAH), but the use of infrared laser ablation (LAH) has become a popular technique due to its' efficiency (time), elimination of acidic media and precise control of zona pellucida opening (size). Fragment removal (FR) of cytoplasmic extrusions can be performed to increase cell-cell contact by eliminating apoptotic tissues within the embryo. Our study aimed to compare the clinical pregnancy outcomes (gestational sac) in patients undergoing FR during the laboratory's transition from using standard AAH to the LAH technique.

The molecular biology of rotaviruses X: intercellular dissemination of rotavirus NSP4 requires glycosylation and is mediated by direct cell-cell contact through cytoplasmic extrusions

The effect of expressing the NSP4 protein of group A rotaviruses in cells has been studied. It led to the rapid appearance of long cytoplasmic extrusions, which, through site-directed mutagenesis of the N-linked glycosylation sites near the amino terminus of the protein, were shown to be dependent on its ability to become fully glycosylated. Real-time confocal microscopy was used to follow the appearance of similar cytoplasmic extrusions in virus-infected cells and revealed them to grow at a rate of ~2 microns/min to more than three cell diameters and to have a lifespan of 30-60 minutes. CellTracker dyes were used to label cell populations and facilitate the monitoring of the transfer of cytoplasm from virus-infected to surrounding uninfected cells through cytoplasmic extrusions that broke down to vesicles, seen both on the surface of and within uninfected cells in mixed cell populations. Staining of these tagged cell mixtures with monospecific antibody to NSP4 revealed the presence of the protein on uninfected cells, suggesting that the cytoplasmic extrusions formed by virus-infected cells facilitates the direct cell-cell spread of NSP4. This direct cell-cell transfer of infected cell material triggered by expression of glycosylated NSP4 in virus-infected cells may contribute to viral pathogenesis and facilitate host invasion by rotaviruses.

In Vitro Ultramorphological Assessment of Apoptosis on CEMss Induced by Linoleic Acid‐Rich Fraction from Typhonium flagelliforme Tuber

The plant Typhonium flagelliforme, commonly known as “rodent tuber” in Malaysia, is often used as a health supplement and traditional remedy for alternative cancer therapies, including leukemia. This study aimed to evaluate in vitro anti-leukemic activity of dichloromethane extract/fraction number 7 (DCM/F7) from T. flagelliforme tuber on human T4 lymphoblastoid (CEMss) cell line. The DCM extract of tuber has been fractionated by column chromatography. The obtained fractions were evaluated for its cytotoxicity toward CEMss cells as well as human primary blood lymphocytes (PBLs). Assessment of apoptosis produced by the most active fraction was evaluated by various microscopic techniques and further confirmation of apoptosis was done by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Phytochemical screening was done by gas chromatography-mass spectrometry (GC-MS). The results shows that 7 out of 12 fractions showed significant cytotoxicity against the selected cell line CEMss, in which fractions DCM/F7, DCM/F11 and DCM/F12 showed exceptional activity with 3, 5 and 6.2 μg ml−1, respectively. Further studies in the non-cancerous PBL exhibited significant selectivity of DCM/F7 compared to other fractions. Cytological observations showed chromatin condensation, cell shrinkage, abnormalities of cristae, membrane blebbing, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double-staining of acridine orange (AO)/propidium iodide (PI), SEM and TEM. In addition, DCM/F7 has increased the cellular DNA breaks on treated cells. GC-MS revealed that DCM/F7 contains linoleic acid, hexadecanoic acid and 9-hexadecanoic acid. The present results indicate that T. flagelliforme possess a valuable anti-leukemic effect and was able to produce distinctive morphological features of cell death that corresponds to apoptosis.

Zerumbone induces apoptosis in T-acute lymphoblastic leukemia cells

Zerumbone (ZER) is a potential anticancer natural compound, isolated from Zingiber zerumbet Smith. In this investigation, the anticancer properties of ZER were evaluated on cancer cells of T-acute lymphoblastic leukemia, CEM-ss. The results showed that ZER has cytotoxic effect against CEM-ss cells with an IC 50 of 8.4 ± 1.9 μg/ml (coefficient of variation < 30%). Comparatively, 5-fluorouracil (positive control), imposed an inhibitory effect on CEM-ss cells with an IC 50 of 1.94 ± 0.06 μg/ml. Scanning electron microscopy (SEM) results revealed abnormalities such as membrane blebbing, holes and cytoplasmic extrusions, all of which are characteristics of apoptosis. In addition, ZER has increased the number of TUNEL-positive stain and the cellular level of caspase-3, the hallmarks of apoptosis, on treated CEM-ss cells. It could be concluded that, ZER was able to produce apoptosis on T-acute lymphoblastic leukemia, CEM-ss. The current findings suggest that ZER might be helpful for improving the usefulness of anticancer agents in the therapy of leukemia.

Electron Microscopic Analysis of Heat-Induced Leakage of Polyphosphate from aphoUMutant ofEscherichia coli

Heat treatment is a simple technique for releasing polyphosphate (polyP) from microbial cells. We investigated the ultrastructural alterations of polyP granules (PPGs) in response to heat treatment at 70 degrees C using a polyP-accumulating phoU mutant of Escherichia coli as a model organism. Electron microscopic and energy-dispersive X-ray analysis suggested concurrent occurrence of PPG degradation and polyP release in the cytoplasm. PolyP is probably released by cytoplasmic extrusion through breaks in the cell envelope.

In Vitro Ultramorphological Assessment of Apoptosis Induced by Zerumbone on (HeLa )

Zerumbone (ZER), a potential anticancer compound, isolated from the fresh rhizomes of Zingiber zerumbet. In this investigation, the cytotoxic properties of ZER were evaluated, on cancer cells of human cervix (HeLa), breast and ovary, and normal cells of Chinese Hamster ovary, using MTT assay. Apoptogenic effects of ZER on HeLa were studied using fluorescence microscopy (AO/PI double staining), scanning and transmission electron microscopy (SEM and TEM), and colorimetric assay of the apoptosis promoter enzyme, caspase-3. The results of MTT assay showed that ZER has less effect on normal cells compared to cancer cells. The lowest IC50 of ZER was observed on HeLa cells. Cytological observations showed nuclear and chromatin condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, holes, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double staining of AO/PI, SEM and TEM. Statistical analysis (two-tailed t-test) of differential counting of 200 cells under fluorescence microscope revealed significant difference in apoptotic cells populations between treated and untreated HeLa cells. In addition, ZER has increased the cellular level of caspase-3 on the treated HeLa cells. It could be concluded that ZER was able to produce distinctive morphological features of cell death that corresponds to apoptosis.

Vitrification of matured bovine oocytes using an intracellular cryoprotectant – trehalose

OBJECTIVE: The technique of oocytes cryopreservation is an important tool in the treatment of infertility associated to assisted reproduction treatments as it can allow the establishment of bank of oocytes for women with cancer or problems with embryo cryopreservation. Attempts to improve the freezing of oocytes have been tested and specially vitrification. The aim of our study was to determine the effectiveness of trehalose as intracellular cryoprotectant in the vitrification of bovine oocytes matured in vitro. DESIGN: Animal, experimental study. MATERIALS AND METHODS: A total of 166 bovine oocytes were included in the study and were allocated in six groups. Three groups were control: TC (vitrification - 10), TP (needle insertion - 10) and TM (trehalose injection - 15) and three groups were submitted to vitrification: TMV (microinjection with trehalose and vitrification - 79), TVDE (ethylenogycol and DMSO and vitrification - 47) and TEV (electropermeabilization with trehalose and vitrification - 15). After two weeks, the oocytes were thawed. The straws containing the oocytes were placed at 41°C in solution one containing TCM 199 with HEPES supplemented with 20% bovine fetal serum and 5mol/l of sucrose for rinsing. For step oocytes were kept in the same solution for 5 minutes. For step 3 oocytes were kept in the same solution with 2.5mol/l of sucrose for 5 minutes and for step 4 oocytes were kept in solution without sucrose for 5 minutes. After thawing we evaluated the cytoplasmic re-expansion capacity, membrane integrity using propidium iodide and glycoprotein alterations of the zona pellucida marked with lectins conjugated with fluorescein 5- isotiocianato (FITC). RESULTS: The evaluation of the return to the isotonic volume after thawing showed a higher rate in group TEV (80%) when compared to group TMV (55%) and TVDE (68%). Oocytes of group TEV presented lower cytoplasmic extrusion (13%). The evaluation of membrane integrity showed membrane damage in 36%, 64% and 29% of the oocytes of the treatment groups TMV, TVDE and TEV, respectively. The presence of manose and sialic acid at the zona pellucida surface, after thawing, did not present difference among treatment groups (100% for all groups). CONCLUSIONS: The obtained results demonstrate that intracellular injection of trehalose has an important crioprotectant effect for vitrification of bovine oocytes matured in vitro, however more studies are necessary in order to minimize cellular damages before its application for human oocytes.