Lactic acid bacteria feeding reversed the malformed eye structures and ameliorated gut microbiota profiles of Drosophila melanogaster Alzheimer's disease model.

AIMS: To utilize transgenic GMR-Aß42 Drosophila melanogaster as a model to evaluate potential Alzheimer's disease (AD)-reversal effects via the administration of lactic acid bacteria (LAB) strains, and associations of LAB with changes in gut microbiota profiles. METHODS AND RESULTS: Wild-type flies (Oregon-R) were crossed with glass multimer reporter-GAL4 (GMR-GAL4) to produce GMR-OreR (Control), while UAS-Aß42 (#33769) were crossed with GMR-GAL4 to produce transgenic Drosophila line that expressed Aß42 (GMR-Aß42). Feed containing seven different LAB strains (Lactobacillus paracasei 0291, Lactobacillus helveticus 1515, Lactobacillus reuteri 30242, L. reuteri 8513d, Lactobacillus fermentum 8312, Lactobacillus casei Y, Lactobacillus sakei Probio65) were given to GMR-Aß42 respectively, while feed without LAB strains were given to control and transgenic GMR-Aß42.nf Drosophila lines. The morphology of the eyes was viewed with scanning electron microscopy (SEM). The changes in gut microbiota profiles associated with LAB were analysed using 16s high throughput sequencing. Malformation of eye structures in transgenic GMR-Aß42 Drosophila were reversed upon the administration of LAB strains, with more prevalent effects from L. sakei Probio65 and L. paracasei 0291. The GMR-Aß42.nf group showed dominance of Wolbachia in the gut, a genus that was almost absent in the normal control group (P < 0·05). The administration of L. sakei Probio65 and L. paracasei 0291 reduced the abundance of Wolbachia accompanied by increased abundance of Stenotrophomonas and Acetobacter (P < 0·05), resembling the microbial profile of the control group. CONCLUSIONS: Lactobacillus sakei Probio65 and Lactobacillus paracasei 0291 have more prominent effects in reversing malformed eye of transgenic GMR-Aß42 Drosophila, and reducing the abundance of Wolbachia accompanied by an increased abundance of Stenotrophomonas and Acetobacter. SIGNIFICANCE AND IMPACT OF THE STUDY: Potentials of LAB to prevent and/or alleviate the onset and pathogenesis of neurodegenerative diseases such as AD, supporting brain health strategies along the gut-brain axis.

Glycoprotein M is important for the efficient incorporation of glycoprotein H-L into herpes simplex virus type 1 particles.

Herpes simplex virus type 1 glycoprotein M (gM) is a type III membrane protein conserved throughout the family Herpesviridae. However, despite this conservation, gM is classed as a non-essential protein in most alphaherpesviruses. Previous data have suggested that gM is involved in secondary envelopment, although how gM functions in this process is unknown. Using transfection-based assays, we have previously shown that gM is able to mediate the internalization and subcellular targeting of other viral envelope proteins, suggesting a possible role for gM in localizing herpesvirus envelope proteins to sites of secondary envelopment. To investigate the role of gM in infected cells, we have now analysed viral envelope protein localization and virion incorporation in cells infected with a gM-deletion virus or its revertant. In the absence of gM expression, we observed a substantial inhibition of glycoprotein H-L (gH-L) internalization from the surface of infected cells. Although deletion of gM does not affect expression of gH and gL, virions assembled in the absence of gM demonstrated significantly reduced levels of gH-L, correlating with defects of the gM-negative virus in entry and cell-to-cell spread. These data suggest an important role of gM in mediating the specific internalization and efficient targeting of gH-L to sites of secondary envelopment in infected cells.

The VC1777-VC1779 proteins are members of a sialic acid-specific subfamily of TRAP transporters (SiaPQM) and constitute the sole route of sialic acid uptake in the human pathogen Vibrio cholerae.

Sialic acids are nine-carbon amino sugars that are present on all mucous membranes and are often used by bacteria as nutrients. In pathogenic Vibrio the genes for sialic acid catabolism (SAC) are known to be important for host colonization, yet the route for sialic acid uptake is not proven. Vibrio cholerae contains a tripartite ATP-independent periplasmic (TRAP) transporter, SiaPQM (VC1777-VC1779), encoded by genes within the Vibrio pathogenicity island-2 (VPI-2), which are adjacent to the SAC genes nanA, nanE and nanK. We demonstrate a correlation of the occurrence of VPI-2 and the ability of Vibrio to grow on the common sialic acid N-acetylneuraminic acid (Neu5Ac), and that a V. cholerae N16961 mutant defective in vc1777, encoding the large membrane protein component of the TRAP transporter, SiaM, is unable to grow on Neu5Ac as the sole carbon source. Using the genome context and known structures of the SiaP protein component of the TRAP transporter, we define a subfamily of Neu5Ac-specific TRAP transporters, of which the vc1777-vc1779 genes are the only representatives in V. cholerae. A recent report has suggested that an entirely different TRAP transporter (VC1927-VC1929) is the Neu5Ac transporter in V. cholerae. Bioinformatics and genomic analysis suggest strongly that this is a C(4)-dicarboxylate-specific TRAP transporter, and indeed disruption of vc1929 results in a defect in growth on C(4)-dicarboxylates but not Neu5Ac. Together these data demonstrate unequivocally that the siaPQM-encoded TRAP transporter within VPI-2 is the sole sialic acid transporter in V. cholerae.

A Survey of Zone II Flexor Tendon Repair Techniques and Rehabilitation Protocols Preferred by Malaysian Orthopaedic Practitioners.

Introduction: Flexor tendon repair is challenging mainly due to the need to balance between a strong repair technique, ease of tendon gliding and early mobilisation to prevent adhesions while preventing tendon rupture. While different countries have different preferences in repair techniques, core sutures and suture types, there is still no study in Malaysia regarding our preference and whether we are following the current evidence. Materials and methods: We performed a survey with a standard questionnaire distributed during our annual national orthopaedic meeting in 2019. The standard questionnaire consisted of 24-objective multiple-choice questions concerning the treatment of flexor tendon injury were distributed with consent. A total of 290 questionnaires that were filled out correctly were included in this study. Results: The majority of respondents preferred the Modified Kessler technique (n=96, 33.1%) followed by the Adelaide technique (n=81, 27.9%) and Double Modified Kessler (n=45, 15.5%). However, for the number of core strands in the repair, the majority utilised the 4-strand (n=203, 70%), followed by 2-strand (n=34, 11.7%) and 6-strand (n=21, 7.2%). The majority utilised Prolene sutures (n=259, 89.3%) with a suture size of 4/0 (n=157, 54.1%). For rehabilitation, 56.9% (n=165) preferred early passive motion, 27.6% (n=80) early active motion and 14.8% (n=43) would strictly immobilise. Conclusion: There is still no consensus as to the best technique; however, the aim of tendon repairs is still the same around the world. It would be helpful to know our preferences to improve our current practice and outcomes following these common flexor tendon injuries in hand.

Evidence for high gene flow, nonrandom mating, and genetic bottlenecks of Ganoderma boninense infecting oil palm (Elaeis guineensis Jacq.) plantations in Malaysia and Indonesia.

Ganoderma boninense, the causal agent of basal stem rot (BSR) disease, has been recognized as a major economic threat to commercial plantings of oil palm (Elaeis guineensis Jacq.) in Southeast Asia, which supplies 86% of the world's palm oil. High genetic diversity and gene flow among regional populations of 417 G. boninense isolates collected from Sabah, Sarawak, and Peninsular Malaysia (Malaysia) and Sumatra (Indonesia) were demonstrated using 16 microsatellite loci. Three genetic clusters and different admixed populations of G. boninense across regions were detected, and they appeared to follow the spread of the fungus from the oldest (Peninsular Malaysia and Sumatra) to younger generations of oil palm plantings (Sabah and Sarawak). Low spatial genetic differentiation of G. boninense (FST = 0.05) among the sampling regions revealed geographically nonrestricted gene dispersal, but isolation by distance was still evident. Analysis of molecular variance (AMOVA) confirmed the little to no genetic differentiation among the pathogen populations and the three genetic clusters defined by STRUCTURE and minimum spanning network. Despite G. boninense being highly outcrossing and spread by sexual spores, linkage disequilibrium was detected in 7 of the 14 populations. Linkage disequilibrium indicated that the reproduction of the fungus was not entirely by random mating and genetic drift could be an important structuring factor. Furthermore, evidence of population bottleneck was indicated in the oldest oil palm plantations as detected in genetic clusters 2 and 3, which consisted mainly of Peninsular Malaysia and Sumatra isolates. The population bottleneck or founding event could have arisen from either new planting or replanting after the removal of large number of palm hosts. The present study also demonstrated that migration and nonrandom mating of G. boninense could be important for survival and adaptation to new palm hosts.

Genetic diversity and gene flow amongst admixed populations of Ganoderma boninense, causal agent of basal stem rot in African oil palm (Elaeis guineensis Jacq.) in Sarawak (Malaysia), Peninsular Malaysia, and Sumatra (Indonesia).

In 1911 and 1917, the first commercial plantings of African oil palm (Elaeis guineensis Jacq.) were made in Indonesia and Malaysia in Southeast Asia. In less than 15 years, basal stem rot (BSR) was reported in Malaysia. It took nearly another seven decades to identify the main causal agent of BSR as the fungus, Ganoderma boninense. Since then, research efforts have focused on understanding G. boninense disease epidemiology, biology, and etiology, but limited progress was made to characterize pathogen genetic diversity, spatial structure, pathogenicity, and virulence. This study describes pathogen variability, gene flow, population differentiation, and genetic structure of G. boninense in Sarawak (Malaysia), Peninsular Malaysia, and Sumatra (Indonesia) inferred by 16 highly polymorphic cDNA-SSR (simple sequence repeat) markers. Marker-inferred genotypic diversity indicated a high level of pathogen variability among individuals within a population and among different populations. This genetic variability is clearly the result of outcrossing between basidiospores to produce recombinant genotypes. Although our results indicated high gene flow among the populations, there was no significant genetic differentiation among G. boninense populations on a regional scale. It suggested that G. boninense genetic makeup is similar across a wide region. Furthermore, our results revealed the existence of three admixed genetic clusters of G. boninense associated with BSR-diseased oil palms sampled throughout Sarawak, Peninsular Malaysia, and Sumatra. We postulate that the population structure is likely a reflection of the high genetic variability of G. boninense populations. This, in turn, could be explained by highly successful outcrossing between basidiospores of G. boninense from Southeast Asia and introduced genetic sources from various regions of the world, as well as regional adaptation of various pathogen genotypes to different palm hosts. Pathogen variability and population structure could be employed to deduce the epidemiology of G. boninense, as well as the implications of plantation cultural practices on BSR disease control in different regions.

Lactobacillus probiotics improved the gut microbiota profile of a Drosophila melanogaster Alzheimer's disease model and alleviated neurodegeneration in the eye.

Alzheimer's disease (AD) is a progressive disease and one of the most common forms of neurodegenerative disorders. Emerging evidence is supporting the use of various strategies that modulate gut microbiota to exert neurological and psychological changes. This includes the utilisation of probiotics as a natural and dietary intervention for brain health. Here, we showed the potential AD-reversal effects of Lactobacillus probiotics through feeding to our Drosophila melanogaster AD model. The administration of Lactobacillus strains was able to rescue the rough eye phenotype (REP) seen in AD-induced Drosophila, with a more prominent effect observed upon the administration of Lactobacillus plantarum DR7 (DR7). Furthermore, we analysed the gut microbiota of the AD-induced Drosophila and found elevated levels of Wolbachia. The administration of DR7 restored the gut microbiota diversity of AD-induced Drosophila with a significant reduction in Wolbachia's relative abundance, accompanied by an increase of Stenotrophomonas and Acetobacter. Through functional predictive analyses, Wolbachia was predicted to be positively correlated with neurodegenerative disorders, such as Parkinson's, Huntington's and Alzheimer's diseases, while Stenotrophomonas was negatively correlated with these neurodegenerative disorders. Altogether, our data exhibited DR7's ability to ameliorate the AD effects in our AD-induced Drosophila. Thus, we propose that Wolbachia be used as a potential biomarker for AD.

Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function.

Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold. Lead inhibitors demonstrated robust, on-target cellular inhibition of MDM2/X leading to activation of the p53 tumor suppressor. Significant improvement to target engagement was observed by increasing valency within a single 10FN3 domain, which has not been demonstrated previously. We further established stable reporter cell lines with tunable expression of EGFP-fused 10FN3 domain inhibitors, and showed their intracellular location to be contingent on target engagement. Importantly, competitive inhibition of MDM2/X by small molecules and cell-penetrating peptides led to a readily observable phenotype, indicating significant potential of the developed platform as a robust tool for cell-based drug screening.

Three repellent gels that contain essential oils from local Malaysian plants against dengue vector.

The essential oils of Litsea elliptica, Piper aduncum, and Piper sarmentosum were prepared as repellents in gel formulation, and their repellent properties against Aedes aegypti were experimentally investigated. The lowest effective doses against adult mosquitoes were 0.8%, 0.5%, and 0.4% for Lit. elliptica, P. sarmentosum and P. aduncum, respectively. In laboratory testing with human subjects, all three gels provided over 90.0% repellency at one hour after application and over 80.0% repellency at four hours, compared with 100% and 95.8% protection after one and four hours, respectively, by DEET. In the field, gels with ED95 concentrations of Lit. elliptica, P. aduncum, and P. sarmentosum essential oils provided 99.3%, 97.5%, and 100% protection, respectively, at two hours. The physical properties and biological stability of the three repellents after storage in hot and cold conditions were also compared. In conclusion, all three gels have the potential for development as repellents against Ae. aegypti.

Trophoblastic debris modifies endothelial cell transcriptome in vitro: a mechanism by which fetal cells might control maternal responses to pregnancy.

The mechanisms by which the fetus induces maternal physiological adaptations to pregnancy are unclear. Cellular debris, shed from the placental syncytiotrophoblast into the maternal blood and phagocytosed by maternal endothelial and immune cells, may be one of these mechanisms. Here we show that trophoblastic debris from normal first trimester placentae induces changes in the transcriptome and proteome of endothelial cells in vitro, which might contribute to the adaptation of the maternal cardiovascular system to pregnancy. Trophoblastic debris also induced endothelial cells to transcribe placenta-specific genes, including the vasodilator hormone CSH1, thereby expanding the effective functional size of the placenta. Our data suggest that the deportation of trophoblastic debris is an important part of the complex network of feto-maternal communication.

kappa-Opioid receptor potentiates apoptosis via a phospholipase C pathway in the CNE2 human epithelial tumor cell line.

The mechanism by which kappa-opioid receptor (kappaor) modulated apoptosis was investigated in CNE2 human epithelial tumor cells. Induction of these cells to undergo apoptosis with staurosporine was associated with a massive increase in intracellular cAMP level. The inhibition of the increase in cAMP partially inhibited apoptosis as evidenced by a reduction of PARP and caspase-3 cleavage. Accordingly, a low but significant level of apoptosis is induced in these cells by the elevation of cAMP through the addition of forskolin and isobutylmethylxanthine. The existence of a cAMP-dependent and a cAMP-independent apoptotic pathway is therefore suggested. Receptor binding studies, RT-PCR experiments and Western blot analysis demonstrated the presence of type 1 kappaor in the CNE2 cells. Stimulation of kappaor in these cells resulted in the production of inositol (1,4,5)-trisphosphate, reduction of cAMP level and a marked enhancement of staurosporine-induced apoptosis. The potentiation of apoptosis by kappaor was prevented by inhibition of phospholipase C but was slightly enhanced by the presence of the active cAMP analogues, 8-CPT-cAMP and dibutyryl-cAMP. These data demonstrate for the first time that the phospholipase C pathway activated by type 1 kappaor expressed by cancer cells is involved in the potentiation of apoptosis.

Optimization of palladium-catalyzed polyene cyclizations: suppression of competing hydride transfer from tertiary amines with Dabco and an unexpected hydride transfer from 1,4-dioxane.

[figure: see text] This paper demonstrates that both 1,2,2,6,6-pentamethylpiperidine (PMP) and 1,4-dioxane can act as hydride donors in palladium-catalyzed polyene cyclizations of 2 and 3. Studies using PMP-d3 and dioxane-d8 either incorporate a deuterium atom into the monosubstituted product or completely inhibit the hydride transfer so that the second ring closure occurs in high yield. Dabco is the best substitute for PMP.

Radionuclide contents in building materials used in Hong Kong.

The radionuclide contents of conventional natural raw building materials, coal ash and slag, and finished building products have been determined using gamma-ray spectrometry. Results of brick measurements in their original geometry and in crushed form are compared. The radioactive concentrations in cement and sand, mostly imported from China, are among the lowest measured. However, due to the high radioactivity of aggregates, composed of granite mainly extracted locally, the mean Ra equivalent activity of concrete is high compared with that in some countries. The radioactivity levels of coal ash and slag in Hong Kong are about the average values in other countries. The incorporation of coal ash and slag in ordinary concrete does not alter the radioactivity significantly.

Vertebral artery thrombosis: a rare presentation of primary polycythaemia.

Primary polycythaemia, also known as polycythaemia vera (PV), is a myeloproliferative neoplasm (MPN) which is associated with arterial and venous thrombosis and which can contribute to significant morbidity and mortality if untreated. Arterial thrombosis accounts for a large proportion of PV-related thrombotic events which may manifest as stroke and myocardial infarction. There is an abundance of literature documenting thrombosis arising in the cerebral vasculature secondary to PV. However, vertebral artery thrombosis associated with PV has not been previously described. We present a case of vertebral artery thrombosis as the presenting manifestation of PV. This case demonstrates the importance of recognising MPNs as a cause of an unusual presentation of thrombosis.

Serological survey of antibodies to influenza A viruses in a group of people without a history of influenza vaccination.

A serological survey for antibodies to influenza viruses was performed in China on a group of people without a history of influenza vaccination. Using the haemagglutination inhibition (HI) assay, we found seropositivity rates for seasonal H3N2 to be significantly higher than those for seasonal H1N1. Samples positive for antibodies to the pandemic (H1N1) 2009 virus increased from 0.6% pre-outbreak to 4.5% (p <0.01) at 1 year post-outbreak. Interestingly, HI and neutralization tests showed that 1.4% of people in the group have antibodies recognizing H9N2 avian influenza viruses, suggesting that infection with this subtype may be more common than previously thought.

A new record of Fannia pusio (Wiedemann) (Diptera:Fanniidae) from Malaysia.

Fannia pusio (Wiedemann) (Diptera: Fanniidae) is newly recorded from Malaysia. This record is based on 1male symbol 1female symbol from Sarawak, east Malaysia and 1male symbol 2female symbol from Selangor, peninsular Malaysia. It is included in the pusio group of Fannia wherein are included Fannia femoralis (Stein), Fannia howardi Malloch, Fannia trimaculata (Stein), Fannia leucosticta (Meigen) and Fannia punctiventris Malloch. The male of Fannia pusio is differentiated from other members of the group by the following features: hind femur with a swelling bearing a number of setae that are usually curled at tip; squamae creamy; tergite 1+2 broadly grey dusted at sides.

Amino acid sequence of chicken gizzard gamma-tropomyosin.

Chicken gizzard muscle tropomyosin has been fractionated into its two major components, beta and gamma and the amino acid sequence of the gamma component established by the isolation and sequence analysis of fragments derived from cyanogen bromide cleavage and tryptic digestions. Despite its much slower mobility on sodium dodecyl sulfate-polyacrylamide electrophoretic gels, it has the same polypeptide chain length (284 residues) as the alpha and beta components of rabbit skeletal muscle. Evidence for microheterogeneity of the chicken gizzard component was detected both on electrophoretic gels and in the sequence analysis. The gamma component is more closely related to rabbit skeletal alpha-tropomyosin than to the beta component. While the protein is highly homologous to the rabbit skeletal tropomyosins, significant sequence differences are observed in two regions; between residues 42-83 and 258-284. In the latter region (COOH-terminal) the alterations in sequence are very similar to those seen in platelet tropomyosin when compared with the skeletal proteins.

Synthesis of a model protein of defined secondary and quaternary structure. Effect of chain length on the stabilization and formation of two-stranded alpha-helical coiled-coils.

Five polyheptapeptides (Ac-(Lys-Leu-Glu-Ala-Leu-Glu-Gly)n-Lys-amide, where n = 1-5) of 8, 15, 22, 29, and 36 residues were synthesized by the solid-phase method. The peptides were purified by reversed-phase high-performance liquid chromatography. The ability of these peptides to form two-stranded alpha-helical coiled-coils in benign medium (1.1 M KC1, 0.05 M PO4 buffer, pH 7.0) was monitored by molecular weight determinations and circular dichroism studies and their physical properties were compared to carboxamidomethylated alpha-tropomyosin at cysteine 190 (CM-tropomyosin). The peptides TM-8, TM-15, and TM-22 were shown to be monomeric in both denaturant (8 M urea) and benign medium by gel-filtration high-performance liquid chromatography on TSK G2000 SW while peptides TM-29 and TM-36 were shown to be dimeric in benign medium both by gel-filtration and sedimentation equilibrium experiments. The CD spectra of the polyheptapeptides TM-8, TM-15, and TM-22 show large increases in molar ellipticity at 220 nm on the addition of trifluoroethanol (helix-inducing solvent) to the benign buffer. By comparison, the two-stranded polyheptapeptides (TM-29 and TM-36) and CM-tropomyosin do not show any increase in molar ellipticity at 220 nm. The helicity of polyheptapeptides increases with increasing chain length, with TM-36 having a value comparable with CM-tropomyosin [( theta]220 = -31,800 degrees and -32,200 degrees, respectively) which is considered to be essentially 100% alpha-helical. These small two-stranded alpha-helical coiled-coils are considerably more stable to temperature and urea denaturation than CM-tropomyosin. Whereas CM-tropomyosin is almost completely denatured in the presence of 6 M urea, TM-29 and TM-36 maintain 22 and 70% of their helicity, respectively. The 30% denaturation values (t30) are 74, 62, and 37 degrees C for TM-36, TM-29, and CM-tropomyosin, respectively, in benign medium (1.1 M KC1:PO4 buffer, pH 7.0). The t30 values can be substantially decreased in the presence of denaturant (3 M urea, 0.1 M KC1, PO4 buffer, pH 7.0) to 62 and 43 degrees C for TM-36 and TM-29, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Cobra venom cardiotoxin induces perturbations of cytosolic calcium homeostasis and hypercontracture in adult rat ventricular myocytes.

The effects of Cobra venom cardiotoxin (CTX) on the cellular morphology, twitch amplitude and intracellular calcium ([Ca2+]i) of the ventricular myocytes were studied. [Ca2+]i and twitch amplitude were determined with a fluorometric ratio method using Fura-2/AM and Calcium Green-1 as calcium indicators, and a videomicroscopic technique, respectively. Addition of 0.001-1 microM CTX led to a time-dependent loss of rod shaped cells, beginning at 1 min, and remaining stable by 20 min. CTX 1 microM initially caused a transient augmentation in amplitude of the electrically induced-[Ca2+]i transient and twitch amplitude in the single cardiac myocyte. This was followed by a prolongation in duration of [Ca2+]i. Eventually, cells became inexcitable and abruptly underwent contracture, and [Ca2+]i remained elevated. In the absence of electrical stimulation, 1 microM CTX induced a Ca2+ spike followed by a sustained elevation of [Ca2+]i, an effect different from that of 40 mm KCl or 10 mm caffeine, which caused a transient elevation in [Ca2+]i. Digital imaging microscopy of Calcium Green-1 fluorescence revealed that the increase in [Ca2+]i was accompanied by changes in cell shape without leakage of fluorescence dye in the early stage after administration of the toxin. In the absence of [Ca2+]o, the initial [Ca2+]i spike was reduced, but the second phase of elevation of [Ca2+]i still occurred. In addition, experiments using Mn2+ quench technique suggested that Ca2+-influx was induced by CTX, and that both ryanodine and thapsigargin, known to deplete Ca2+ from its intracellular pool, abolished the second phase of the elevation of [Ca2+]i. The effects of cardiotoxin were abolished by 10 mM Ni2+ and 10 mM -Ca2+-o, but not by 5 microM verapamil. In conclusion, the observations indicate that CTX causes an initial increase followed by a second sustained elevation in [Ca2+]i, which is accompanied by changes in cell shape-from rod to round-and hypercontracture. The initial [Ca2+]i spikes were attributed to the extracellular Ca2+ influx, while the second [Ca2+]i elevation was related to internal Ca2+ release. The high [Ca2+]i may be responsible for hypercontracture and cell death. Further studies are needed to verify it.