Autodelineation of organ at risk in head and neck cancer radiotherapy using artificial intelligence.

Aim: The aim of this study is to check the practical feasibility of artificial intelligence for day-to-day operations and how it generalizes when the data have considerable interobserver variability. Background: Automated delineation of organ at risk (OAR) using a deep learning model is reasonably accurate. This will considerably reduce the medical professional time in manually contouring the OAR and also reduce the interobserver variation among radiation oncologists. It allows for quick radiation planning which helps in adaptive radiotherapy planning. Materials and Methods: Head and neck (HN) computed tomography (CT) scan data of 113 patients were used in this study. CT scan was done as per the institute protocol. Each patient had about 100-300 slices in Dicom format. A total number of 19,240 images were used as the data set. The OARs were delineated by the radiation oncologist in the contouring system. Of the 113 patient records, 13 records were kept aside as test dataset and the remaining 100 records were used for training the UNet 2D model. The study was performed on the spinal cord and left and right parotids as OARs on HN CT images. The model performance was quantified using the Dice similarity coefficient (DSC) score. Results: The trained model is used to predict three OARs, spinal cord and left and right parotids. The DSC score of 84% and above could be achieved using the UNet 2D Convolutional Neural Network. Conclusion: This study showed that the accuracy of predicted organs was within acceptable DSC scores, even when the underlying dataset has significant interobserver variability.

Isolation and characterization of novel degradation products of Doxofylline using HPLC, FTIR, LCMS and NMR.

Forced degradation of Doxofylline (DFL) in different stress (base and peroxide) conditions gave rise to two potential unknown impurities. These unknown degradation products DFL DEG-I and DFL DEG-II were evaluated using a new-reverse-phase high performance liquid chromatography (HPLC), where it was eluted at 0.44 and 1.09 relative retention times to DFL peak. DFL DEG-I and DFL DEG-II were isolated using preparative HPLC from degradation mixtures. The structure of DFL DEG-I and DFL DEG-II were elucidated using high resolution MS, multi-dimensional NMR and FTIR spectroscopic techniques, and characterized. The stereochemistry of the enantiomers in DFL DEG-II has further been investigated using computational techniques. To the best of our knowledge, DFL DEG-I and DFL DEG-II are novel impurities and not reported elsewhere.

Total activity of glutathione-S-transferase (GST) and polymorphisms of GSTM1 and GSTT1 genes conferring risk for the development of age related cataracts.

The pathogenesis of cataract is influenced by a number of factors including oxidative stress. Glutathione-S-transferase (GST) catalyses the nucleophilic addition of the thiol of GSH to electrophilic acceptors. It is important for detoxification of xenobiotics in order to protect tissues from oxidative damage. In humans, GSTT1 and GSTM1 deletion genotypes are associated with a variety of pathological conditions including certain ophthalmic diseases. In the present study, it is aimed to determine the risk of genetic polymorphisms of GSTM1 and GSTT1 isoforms of GST for developing of age related cataracts (ARCs). We compared the prevalence of GSTT1 and GSTM1 deletion genotypes, which were determined by multiplex polymerase chain reaction, in 455 patients with ARCs (108 with nuclear (NC), 105 with cortical (CC), 96 with posterior subcapsular, (PSC) and 146 with mixed type (MT)) and 205 age and sex matched controls. The GST activity in erythrocytes (RBC) and cataractous lenses was measured spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The frequency of GSTM1 positive individuals was significantly higher in MT cataracts followed by NC, CC and PSC types with corresponding decrease in the GSTM1 null genotypes as compared to controls. Considering the GSTT1 locus, GSTT1 null genotypes showed high frequency in patients in general as compared to controls with corresponding reduction in the GSTT1 positive genotype. The activity of GST in RBC was higher in all the types of cataracts as compared to that in controls and in cataractous lenses the mean values were slightly higher in cases of NC cataracts as compared to CC, PSC and MT. The data suggests that GSTM1 positive, GSTT1 null and double null (GSTM1 null and GSTT1 null) genotypes may confer risk for the development of ARC. The increased activity of GST found in the present study could be due to a compensatory mechanism operating in response to increased oxidative stress.

Microtubule turnover in ooplasm biopsy reflects ageing phenomena in the parent oocyte.

Oviductal oocytes retrieved from superovulated B6D2F1 mice at 13.5, 16 and 19 h after human chorionic gonadotrophin (HCG) (groups A, B and C respectively, n = 382) were micromanipulated to obtain 12-20 mum sized ooplasm biopsy fragments. Experiments were divided into three sets. Ooplasmic microtubule dynamics were studied in ooplasm biopsy specimens and parent oocytes (set 1) and ooplasm biopsy specimens (set 2), whilst zona pellucida dissolution time, cortical granule loss and spindle/chromatin morphology using confocal microscopy were also studied in parent oocytes (set 2). Oocytes withstood oocyte biopsy with a high survival rate (98.2%) and the biopsied oocytes underwent successful fertilization and development (set 3). An absolute one-to-one correlation was seen between the oocyte biopsy specimens and the parent oocytes in terms of ooplasmic microtubule dynamics (set 1), and increased ooplasmic microtubule dynamics in oocyte biopsy specimens paralleled ageing phenomena in the parent oocytes (set 2). Zona pellucida dissolution time was significantly lower in parent oocytes from group A versus groups B (P = 0.032), and C (P < 0.001). (Groups A, B, C include minimal, moderate, increased ooplasmic microtubule dynamics in oocyte biopsy specimens respectively.) Oocyte cortical granule loss and spindle/chromatin abnormalities were mainly seen in group C (P < 0.001). Oocyte biopsy can thus be applied to judge age-related changes in the parent oocytes.

Synthesis and biological evaluation of thiophene [3,2-b] pyrrole derivatives as potential anti-inflammatory agents.

A series of thiophene [3,2-b] pyrrole derivatives were synthesized and evaluated their abilities to inhibit anti-inflammatory activity. In this series, substituent effects at the N-1, 2 and 5 positions of thiophene [3,2-b] pyrrole were examined. The results obtained are compared to those previously reported anti-inflammatory drugs like Tenidap sodium, Diclofenac sodium and Piroxicam. The results indicated the critical role of the group linked in the N-1 position and 2, 5 positions of thiophene [3,2-b] pyrrole with different functional groups.

Inositol 1,4,5-trisphosphate receptor function in human oocytes: calcium responses and oocyte activation-related phenomena induced by photolytic release of InsP(3) are blocked by a specific antibody to the type I receptor.

Type I inositol 1,4,5-trisphosphate-sensitive receptors (InsP(3)R) are expressed in human oocytes and may be involved in operating the Ca(2+) release triggered by the fertilizing sperm. This study examines the contribution of type I InsP(3)R in operating Ca(2+) release in human oocytes secondary to InsP(3) itself, using a specific function-blocking antibody in conjunction with photolytic release of microinjected InsP(3). Intracellular Ca(2+) responses were assessed in oocytes microinjected with only caged InsP(3) in experiment set A, while in experiment sets B and C, sibling oocytes were injected with caged InsP(3) and the blocking antibody or a corresponding volume of medium, prior to flash photolysis. In experiment set C, certain fertilization-related phenomena (cortical granule exocytosis and chromatin configurations) were assessed using optical sections and three-dimensional image reconstructions obtained from a confocal laser scanning microscope. In experiment set A, photolytic release of InsP(3) triggered a Ca(2+) response (increase from approximately 100 to 220 nmol/l followed by an exponential recovery, n = 8) and a wave in the oocytes that spread from the stimulation point to the opposite pole. In set B, photolytic InsP(3) release generated Ca(2+) responses in control oocytes (n = 9), but not in the antibody-injected oocytes (n = 7). In set C, cortical granule exocytosis and anaphase chromosome configurations were noted in the control oocytes after flash photolysis (n = 6). These changes were completely absent in antibody injected oocytes as their cortical granules were intact and the chromosomes were in metaphase. These oocytes had also lacked Ca(2+) responses as in set B (n = 5). This study demonstrates the functional presence of type I InsP(3)R-operated Ca(2+) channels in human oocytes and further suggests an active role of InsP(3) in triggering the Ca(2+) rise and secondary activation phenomena at fertilization.

Cryopreservation of human germinal vesicle stage and in vitro matured M II oocytes: influence of cryopreservation media on the survival, fertilization, and early cleavage divisions.

OBJECTIVE: To study the influence of low-sodium cryopreservation media (CPM) on the survival and development of frozen-thawed germinal vesicle (GV) stage and in vitro matured human oocytes. DESIGN: Prospective experimental study. SETTING: Academic hospital-based fertility center. PATIENT(S): Experimental groups: Oocytes cryopreserved at the GV (group A, n = 63 and group B, n = 64) or M II stage (group C, n = 62) with use of conventional (group A) or low-sodium CPM (groups B and C). Control groups: Sibling GV stage oocytes subjected to in vitro maturation (IVM; control group A, n = 64; control group B, n = 64). INTERVENTION(S): IVM, intracytoplasmic sperm injection and subsequent culture. MAIN OUTCOME MEASURE(S): Rates of survival, maturation, fertilization, and cleavage. RESULT(S): The postthaw survival was significantly lower in groups A (57.1%) and B (48.4%) compared to C (84.4%). In group A, maturation and cleavage rates were significantly lower, and fertilization rate was similar to controls (GVBD: 72.2% vs. 90.6%; progression to M II: 33.3% vs. 76.6%; cleavage: 42.9% vs. 88.2%; and fertilization: 58.3% vs. 69.4% in group A vs. control group A, respectively). There was no such difference in group B. In group C, despite a slight but significant lowering of the rate of 2 PN and an increase in that of 3 PN (2 PN: 47.4% vs. 70.2% and 3 PN: 15.8% vs. 3.2% in group C vs. total controls, respectively), embryonic cleavage per GV oocyte was significantly higher (25.8%) compared to group A (4.8%) but not to group B (15.6%). The rate of maturation and cleavage per surviving GV oocyte was significantly higher in group B than group A. CONCLUSION(S): Low-sodium-based CPM is beneficial for in vitro matured M II stage oocytes and is significantly better than the conventional sodium-based media for the GV stage oocytes.

Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity.

OBJECTIVE: To study the effect of delayed intracytoplasmic sperm injection (ICSI) on the fertilization and cleavage of human in vitro matured (IVM) oocytes. DESIGN: Prospective experimental study. SETTING: Academic hospital-based fertility center. PATIENT(S): The experimental group consisted of 73 spare germinal vesicle-stage oocytes from 25 patients. The control group consisted of sibling in vivo matured oocytes from the same patients that were subjected to ICSI in the clinical program. INTERVENTION(S): Equal numbers of sibling IVM oocytes were subjected to ICSI either soon after maturation (30 hours, group 1) or after a 6-hour delay (36 hours, group 2). In a subsequent set of experiments, spermatozoa were labeled with a fluorescent mitochondria-specific vital dye and injected into 17 IVM oocytes that were intentionally aged by 6-8 hours. The resultant zygotes were fixed. MAIN OUTCOME MEASURE(S): Incidence of fertilization and cleavage, numbers and mean diameters of pronuclei, and incidence of zygotes with significant pronucleus size asynchrony. Identification of the male pronucleus by its proximity to the fluorescent sperm midpiece remnant. RESULT(S): Group 2 had significantly lower rates of normal fertilization (60%) than the control group (82.9%) and significantly lower cleavage rates (46.7%) than both group 1 (85%) and the control group (98.1%). The incidence of oocytes that developed one pronucleus and pronucleus size asynchrony was significantly higher in group 2 (32% and 40%, respectively) than in group 1 (4% and 5%, respectively) and in the control group (4.1% and 4.4%, respectively). All the zygotes with significant pronucleus size asynchrony that developed after delayed ICSI with labeled spermatozoa showed proximity of the fluorescent sperm midpiece remnant to the smaller pronucleus. CONCLUSION(S): For IVM oocytes, the incidence of one pronucleus, pronucleus size asynchrony (possibly related to a smaller male pronucleus), and cleavage failure increase when ICSI is delayed after maturation. Thus, the timing of ICSI is critical for optimum fertilization of IVM oocytes.

Presence and dynamic redistribution of type I inositol 1,4,5-trisphosphate receptors in human oocytes and embryos during in-vitro maturation, fertilization and early cleavage divisions.

We studied the presence and distribution of the intracellular calcium channel regulating type I inositol 1,4,5-trisphosphate receptors (IP3R) in human immature and mature oocytes, pronuclear zygotes and cleaved embryos using a specific antibody. Two approaches were used: (i) fluorescence immunocytochemistry using a confocal laser scanning microscope (CLSM) and (ii) Western blotting. With confocal microscopy, the receptors were found in the oocytes, fertilized zygotes as well as cleaved embryos at all stages studied. The pattern and distribution of the receptor staining in the oocytes changed gradually from a diffuse granular patchy one at the germinal vesicle (GV) stage to a reticular and predominantly peripheral one through the metaphase I and metaphase II (MII) stages. After fertilization, the distribution changed gradually to both, peripheral and central in the zygotes and early 2-4-cell embryos and predominantly perinuclear in the 6-8-cell embryos. Furthermore, an overall increase in the staining intensity was observed from GV to MII stage oocytes and from zygotes to 6-8-cell embryos. We also studied the spatial distribution of the receptor in detail by constructing three-dimensional images from the serial optical sections obtained on the CLSM. Peculiar peripheral aggregates of receptor clusters were noted in the MII stage oocytes, zygotes and some blastomeres from early cleaved embryos. Finally, Western blots performed on the extracts of 72 in-vitro matured oocytes and 50 spare cleavage stage embryos showed positive bands at approximately 260 kDa. These findings coincide with and thus possibly represent the dynamic changes occurring in the cellular Ca2+ release systems through oocyte maturation, fertilization and early embryogenesis. Thus, type I IP3R are likely to play a role during these stages of early development in the human.

Effect of post-ovulatory age and calcium in the injection medium on the male pronucleus formation and metaphase entry following injection of human spermatozoa into golden hamster oocytes.

The occurrence of parthenogenetic activation is a major hurdle in obtaining sperm chromosome metaphases after heterospecific intracytoplasmic sperm injection (ICSI) of golden hamster oocytes with human spermatozoa. We addressed two potential contributors to parthenogenetic activation namely, post-ovulatory age of the oocyte and Ca2+ content of the injection medium. In serial experiments, hamster oocytes were retrieved at 11.5, 13, 16 and 21 h after the ovulatory dose of human chorionic gonadotrophin (HCG) and microinjected with human spermatozoa suspended alternately in a regular (1.9 mM Ca2+) or a Ca2+-free medium. A progressive decrease in the rates of male pronucleus (MPN) formation and metaphase entry and increase in the rates of parthenogenetic activation without male pronucleus occurred with increasing post-ovulatory age. The favourable influence of Ca2+-free injection medium on the mean rates of MPN and metaphase entry was restricted to the relatively older oocytes (MPN 16 h: 49.5 versus 32.3%, P< 0.008; 21 h: 22.2 versus 11.1%, P< 0.001; metaphase entry 16 h: 36.8 versus 25.1%, P< 0.02; 21 h: 13.3 versus 5.2%, P< 0.01 in the Ca2+-free and regular groups respectively). Our data confirm the increased activation sensitivity with post-ovulatory ageing and its adverse influence on the MPN formation and metaphase entry after heterospecific ICSI of hamster oocytes.

In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium.

In-vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side-effects of gonadotrophin stimulation for in-vitro fertilization (IVF). The pregnancy rates from oocytes matured in vitro are much lower than those of in-vivo stimulation cycles indicating that optimization of IVM remains a challenge. Therefore, we investigated the effect of supplementation of the medium with gonadotrophins, oestradiol and epidermal growth factor (EGF) and the effect of retaining or removing the cumulus cells on nuclear and cytoplasmic maturation of immature oocytes. Human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation for intracytoplasmic sperm injection (ICSI) were cultured in a complex defined medium either supplemented with gonadotrophins, oestradiol and physiological concentrations of EGF (2 ng/ml) or gonadotrophins and oestradiol alone. The cumulus cells were either removed or kept intact. In GV stage oocytes cultured without cumulus (group I) significantly more oocytes reached the metaphase II (MII) stage at 30 h in media supplemented with EGF (64.3 versus 33.9%, P < 0.003). For oocytes cultured with intact cumulus (group II), more oocytes reached MII at 30 h than in group I, but there was no difference in medium with or without EGF supplementation (81.8 and 79.8% respectively). Cytoplasmic maturation of MII oocytes was judged from their capability to activate and fertilize after ICSI. In group I, the rates of activation and normal fertilization were similar. However, in group II, significantly more oocytes underwent normal fertilization in the EGF-supplemented than the unsupplemented group (71.7 versus 45.6%, P < 0.05). The cleavage rates of the fertilized oocytes were similar in the sibling oocyte subgroups cultured with or without EGF supplementation, but the overall cleavage rates were higher in cumulus-intact compared to cumulus-denuded oocytes (88.9 versus 47.8%, P < 0.001). Thus, supplementation of the maturation medium with EGF and maintenance of the cumulus during culture improve the nuclear and cytoplasmic maturation of human oocytes in vitro.

Chromatin decondensation, pronucleus formation, metaphase entry and chromosome complements of human spermatozoa after intracytoplasmic sperm injection into hamster oocytes.

Obtaining karyotypes from human spermatozoa after microinjection into Syrian golden hamster oocytes is difficult and the hitherto reported results are unsatisfactory. This may be related to the injection and culture technique or to the high susceptibility of the hamster oocytes to undergo parthenogenetic activation or both. Therefore, we investigated the hamster oocyte-human sperm microinjection model using the following two approaches: (i) application of contemporary techniques for injection (touching the sperm tail) and culture (hamster embryo culture medium, HECM-3, 10% CO2) and (ii) omission of Ca2+ from the injection medium. Thus, in the first series of experiments, 252 hamster oocytes were injected with human spermatozoa. Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronucleus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had developed only the hamster female PN while the sperm nuclei were either intact or swollen (partially decondensed), indicating that failure of oocyte activation was not the likely reason for the failure of male PN formation in these oocytes. In the next series of experiments, sibling oocytes were alternately injected with spermatozoa suspended either in the regular (1.9 mM Ca2+) or Ca2+-free injection medium (experiment set 2, n=278). A significant improvement was noted in the mean percentages of oocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in the Ca2+-free group versus the regular group (2PN, 2PB: 51 versus 36.6%, metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus 20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be one of the contributing factors for the failure of male PN formation after heterospecific hamster ICSI. From these experiments it can be concluded that application of the advanced injection and culture techniques and omission of Ca2+ from the injection medium are promising for the routine application of the hamster oocyte microinjection for karyotyping of human spermatozoa with poor fertilizing capacity.

Power loss, modal noise and distortion due to microbending of optical fibers.

Theoretical and experimental investigations are described for determining the transmission characteristics of a multimode fiber with microbending for coherent and partially coherent illumination. The measured values of the average excess power loss are shown to be in close agreement with the theory. Also, an estimate of the excess transient loss due to mode coupling is found to be in good agreement with previously published data. Mode-mode interference is shown to be the cause of temporal fluctuations in the microbending loss, from which expressions for modal noise and baseband/subcarrier nonlinearity are derived on a statistical basis. For a given overall loss, the results show that many uniformly distributed small amplitude microbends cause much less modal noise and distortion than a few large amplitude microbends.

Microbending dependence of phase in single-mode fibers.

The dependence of the radio-frequency phase shift on microbending loss for single-mode fibers has been investigated theoretically. Using representative source and fiber cable parameter values, the frequency stability of a prototype fiber link is found to be about 10(-16) for an averaging time of 10(3) sec.

Optimized groove profiles for diffraction gratings obtained by an analytical method.

It is shown that diffraction gratings of arbitrary groove profile can be analyzed analytically. The analytical method is then used to obtain an optimized groove profile.