Comparative cryopreservation of indian wild orange (Citrus indica Tanaka) embryonic axes.

BACKGROUND: Indian Wild Orange (Citrus indica Tanaka) is an endangered and endemic species from northeast India for which effective ex situ conservation strategies, including embryo cryopreservation, are urgently needed. MATERIALS AND METHODS: Desiccation tolerance and cryopreservation ability for embryonic axes of Citrus indica was determined using three techniques (air desiccation-freezing, PVS2 vitrification-freezing and encapsulation-dehydration-freezing). Success was assessed as survival and recovery in vitro. RESULTS: Successful cryopreservation of embryonic axes was achieved using all three methods, with the highest survival achieved when using air desiccation-freezing (90%) followed by encapsulation-dehydration (85%) and PVS2 vitrification cryopreservation (80%). Regeneration levels were lower than survival levels for all three proceedures. Post-cryo regeneration success was: encapsulation-dehydration (64%) > air desiccation-freezing (55%) > PVS2 vitrification (52%). CONCLUSION: Although there was relatively high post-cryopreservation recovery growth obtained using all the three techniques, the air desiccation-freezing technique is preferred, as it is a simple, practical and reproducible technique for the long-term cryobanking of this important wild species. Doi: 10.54680/fr23310110512.

Storage Behavior and Cryopreservation of Citrus cavaleriei, an Endangered, Cold-resistant Species of Northeast India with Exceptionally Large Seeds.

BACKGROUND: Ichang Papeda (Citrus cavaleriei H.Lév. ex Cavalerie) is a wild and endangered species of NE India that requires urgent preservation of its genetic resources. OBJECTIVE: To characterize the storage physiology of the seeds and to cryopreserve the embryo and embryonic axis (EA). MATERIALS AND METHODS: The morphology and storage physiology of the seeds were determined, and the cryopreservation of embryos and EA attempted using various techniques (viz., air desiccation-freezing, vitrification and encapsulation-dehydration). RESULTS: Weighing up to 4 g, seeds of Citrus cavaleriei are the largest known in the genus. Based on estimates using the seed coat ratio - seed mass (SCR-SM) model there was a very high probability of seed desiccation intolerance, which was validated physiologically; seeds lost vigour on drying below 30% moisture content (MC) and no seeds germinating after drying to <12% MC. Embryos and EAs could be air dried to 25-30% MC and cryopreserved with c. 50% survival. In contrast, EA optimally exposed to PVS2 (20 min) or encapsulated, sucrose pretreated (0.5 M, 24 h) and dehydrated (6 h) had c. 40% survival after cryopreservation. CONCLUSION: Citrus cavaleriei produces large, recalcitrant seeds that can, nevertheless, be cryopreserved as embryos or isolated EA after air drying to c. 25-30% MC; encapsulation-dehydration and vitrification provide alternative options for the cryopreservation of EA.

Mycorrhization in trees: ecology, physiology, emerging technologies and beyond.

Mycorrhization has been an integral part of plants since colonization by the early land plants. Over decades, substantial research has highlighted its potential role in improving nutritional efficiency and growth, development and survival of crop plants. However, the focus of this review is trees. Evidence have been provided to explain ecological and physiological significance of mycorrhization in trees. Advances in recent technologies (e.g., metagenomics, artificial intelligence, machine learning, agricultural drones) may open new windows to apply this knowledge in promoting tree growth in forest ecosystems. Dual mycorrhization relationships in trees and even triple relationships among trees, mycorrhizal fungi and bacteria offer an interesting physiological system to understand how plants interact with other organisms for better survival. Besides, studies indicate additional roles of mycorrhization in learning, memorizing and communication between host trees through a common mycorrhizal network (CMN). Recent observations in trees suggest that mycorrhization may even promote tolerance to multiple abiotic (e.g., drought, salt, heavy metal stress) and biotic (e.g. fungi) stresses. Due to the extent of physiological reliance, local adaptation of trees is heavily impacted by the mycorrhizal community. This knowledge opens the possibility of a non-GMO avenue to promote tree growth and development. Indeed, mycorrhization could impact growth of trees in nurserys and subsequent survival of the inoculated trees in field conditions. Future studies might integrate hyperspectral imaging and drone technologies to identify tree communities that are deficient in nitrogen and spray mycorrhizal spore formulations on them.

Long-term, large scale banking of citrus species embryos: comparisons between cryopreservation and other seed banking temperatures.

The long-term, large scale application of embryo cryopreservation has been assessed rarely and comparisons of viability loss for partially dried material with conventional seed bank storage conditions infrequently made. Five citrus species were cryopreserved following air drying of embryos (seed minus the testa) and embryonic axes: rough lemon (Citrus jambhiri), pommelo (C. grandis), mandarin (C. reticulata), citron (C. medica) and kagzi lime (C. aurantifolia). Although drying rates to c. 10 percent moisture content (MC) were approximately 10-times faster for isolated axes compared to embryos, the optimum MCs for cryopreservation were generally similar within a species, varying from c. 10 percent (C. jambhiri) to c. 20 percent (C. medica). Nonetheless, the hydration window for cryopreservation of the axis was usually wider than for the embryo. For all species, embryo or axis survival after cryopreservation ranged from 65 to 96 percent (C. medica axes), producing normal healthy seedlings from embryos and plantlets from axes without intervening callus growth in vitro. Whilst partially dried embryos of all five species survived fully liquid nitrogen vapour storage for 120 days, viability loss was rapid at -20 degree C, 5 degree C and ambient temperature, with a maximum interpolated half-life across these temperatures of c. 80 days for C. grandis at 5 degree C. The developed cryopreservation protocols were applied routinely to cryobank 377 accessions of Citrus germplasm from field genebanks, farmer's orchards, semi-wild and wild sources. After an average of 6.3 to 8.4 years cryo-storage, between 69 and 81 percent of accessions per species retained > 70 percent of the viability after desiccation. The results provide irrevocable evidence for the importance of cryopreservation for the banking of seeds of higher plants.

Addiction severity index composite scores as predictors for sexual-risk behaviors and drug-use behaviors in drug-using pregnant patients.

BACKGROUND: HIV sexual-risk and drug-use behavior predictors have been studied in non-pregnant but not pregnant drug-dependent populations. OBJECTIVE: Examine the ability of the ASI composite scores to predict HIV sexual- and drug-risk scores as well as the individual items of a modified version of the Risk Assessment Battery in drug-using pregnant women. METHODS: Pregnant women (N = 76) completing pretreatment ASI and HIV-risk questionnaires. RESULTS: The Legal composite score was the sole significant predictor of the sexual-risk score, with a 1 SD increase in the Legal composite score resulting in a 24% increase in sexual-risk, p < .001. The Medical, Drug, and Legal composite scores were each significant predictors of the drug-risk score, with a 1 SD increase resulting in a 31% decrease, and 121% and 73% increases, respectively, in drug-risk, all ps < .05. CONCLUSIONS AND SCIENTIFIC SIGNIFICANCE: Drug-using pregnant women and their fetuses are vulnerable to the consequences of both sexual-risk behaviors and drug-use. The ASI may help screen such patients for HIV sexual-risk and drug-use behaviors as a first step in tailoring treatment to address these issues.

Methodology for surveillance of antimicrobials use among out-patients in Delhi.

BACKGROUND & OBJECTIVE: Determining antibiotic use in the population is problematic in India, as there are no population level databases on prescriptions and over-the-counter sales. This study attempts to establish a methodology for surveillance of antibiotic use in the community. METHODS: Antibiotic use was monitored in fi ve municipal wards of Delhi, from January-December 2004. Thirty co-operative retail pharmacies were enrolled in Inderpuri (7), Karol Bagh (3), Patel Nagar (5), Rajinder Nagar (6) and Rajouri Garden (9); data on antibiotic use were collected in two ways. Firstly, bulk purchase data were collected by recording the quantities of all antibiotics purchased by these pharmacies every month over one year. Secondly, 15-25 'exit interviews' were conducted with patients leaving the enrolled pharmacies every month. Antibiotic use from bulk purchase data was measured as defined daily dose (DDD)/1000 population and from exit interview data as DDD/1000 patients visiting the pharmacy during data collection and also as per cent patients receiving an antibiotic. RESULTS: Bulk purchase and exit interview data showed some similar patterns of antibiotic use with a growing peak in the consumption of most antibiotics classes in the months of February and March. Use of the fluoroquinolone group (J01MA) was much higher than other antibiotic classes by both methods. Exit interviews revealed that 21 per cent of persons who visited the pharmacies bought antibiotics. Both measures of use from exiting patient interviews showed the same trend over time. INTERPRETATION & CONCLUSION: Both drug use methods can be used to measure antibiotic use in the private retail pharmacies and outpatient department of public facilities. Surveillance of antimicrobial drug use from private retail pharmacies in a municipal zone in Delhi indicated overuse of antimicrobial drugs, particularly fluoroquinolines.

Two mouse retinal degenerations caused by missense mutations in the beta-subunit of rod cGMP phosphodiesterase gene.

We report the chromosomal localization, mutant gene identification, ophthalmic appearance, histology, and functional analysis of two new hereditary mouse models of retinal degeneration not having the Pde6brd1("r", "rd", or "rodless") mutation. One strain harbors an autosomal recessive mutation that maps to mouse chromosome 5. Sequence analysis showed that the retinal degeneration is caused by a missense point mutation in exon 13 of the beta-subunit of the rod cGMP phosphodiesterase (beta-PDE) gene (Pde6b). The gene symbol for this strain was set as Pde6brd10, abbreviated rd10 hereafter. Mice homozygous for the rd10 mutation showed histological changes at postnatal day 16 (P16) of age and sclerotic retinal vessels at four weeks of age, consistent with retinal degeneration. Retinal sections were highly positive for TUNEL and activated caspase-3 immunoreactivity, specifically in the outer nuclear layer (ONL). ERGs were never normal, but rod and cone ERG a- and b-waves were easily measured at P18 and steadily declined over 90% by two months of age. Protein extracts from rd10 retinas were positive for beta-PDE immunoreactivity starting at about the same time as wild-type (P10), though signal averaged less than 40% of wild-type. Interestingly, rearing rd10 mice in total darkness delayed degeneration for at least a week, after which morphological and functional loss progressed irregularly. With the second strain, a complementation test with rd1 mice revealed that the retinal degeneration phenotype observed represents a possible new allele of Pde6b. Sequencing demonstrated a missense point mutation in exon 16 of the beta-subunit of rod phosphodiesterase gene, different from the point mutations in rd1 and rd10. The gene symbol for this strain was set as Pde6bnmf137, abbreviated nmf137 hereafter. Mice homozygous for this mutation showed retinal degeneration with a mottled retina and white retinal vessels at three weeks of age. The exon 13 missense mutation (rd10) is the first known occurrence of a second mutant allele spontaneously arising in the Pde6b gene in mice and may provide a model for studying the pathogenesis of autosomal recessive retinitis pigmentosa (arRP) in humans. It may also provide a better model for experimental pharmaceutical-based therapy for RP because of its later onset and milder retinal degeneration than rd1 and nmf137.

Regulatory developments in the conduct of clinical trials in India.

There has been a drop in clinical research in India following stringent conditions put in place by the Indian Supreme Court in 2013. The Court's orders came in the wake of irregularities highlighted in the conduct of clinical trials in the country. This paper highlights the steps taken by the Indian regulator, the Central Drugs Standard Control Organisation to comply with these directions. These are of three kinds: strengthening regulatory institutions, protecting participant safety and creating regulatory certainty for sponsors and investigators. Examples include the large-scale training of Ethics Committees, framing detailed guidelines on compensation and audiovisual recording of the informed consent process, as well as reducing the time taken to process applications. It is expected that these measures will inspire confidence for the much-needed resumption of clinical research.

The release of prolactin by medroxy-progesterone acetate in human subjects.

Medroxyprogesterone acetate an injectable contraceptive when administered to four lactating women at a dose of 150 mg every three months significantly raised prolactin levels over those observed in four control women. Prolactin levels were higher at all times during 4-17 weeks in the drug-treated women, irrespective of whether the samples were collected before or after suckling. It was also observed that while suckling clearly released prolactin in both groups the difference was greater in the women receiving the injection, indicating that the drug enhanced the release of prolactin in response to the suckling stimulus.

The effect of D-norgestrel, 30 micrograms, on the oral glucose tolerance test, including insulin levels, in Thai women.

The effect of D-norgestrel, 30 mug, on the oral glucose tolerance test was studied in 49 Thai women. There was a significant elevation of the blood glucose level at 60 minutes during the test in women who had taken D-norgestrel for 6 and 12 months. Insulin levels in the blood were significantly elevated over control levels in both groups of women at 90, 120, 150, and 180 minutes during the test. There was no difference in the results obtained at 6 months and 12 months. There was also no significant difference in the fasting blood glucose or insulin levels in the three groups of women. The results indicate that D-norestrel at a daily dose of 30 mug has an effect on carbohydrate metabolism in Thai women.

PIP: Oral glucose tolerance tests were performed on 49 Thai women to inve stigate the effect of d-norgestrel (30 mcg) on carbohydrate metabolism. At 60 minutes, women who had taken d-norgestrel for 6 and 12 months showed a significant increase in blood glucose levels (p less than .05). There was a significant increase in blood insulin levels at 90, 120, 150, and 180 minutes in both groups (p less than .05). However, fa sting blood glucose or insulin levels were not markedly different. It is concluded that d-norgestrel, at the dose studied, affects carbohydrat e metabolism in Thai women, and it is suggested that this may be a characteristic effect of 19-nor steroids.

Current status of intrauterine devices. II. Intrauterine devices and pelvic inflammatory disease and ectopic pregnancy.

PIP: The suspected causal relationship between use of different types of IUDs and pelvic inflammatory disease and ectopic pregnancy are reviewed. The information available at this time regarding the IUD and pelvic inflammatory disease (PID) in the early IUDs, the Lippes loop and other polyethylene devices, the copper-containing devices, and the progesterone-containing devices. Grafenberg, in 1929, reported 17 cases of inflammation following insertion of silk IUDs which had been placed entirely within the uterine cavity. He found no such evidence following insertion of 150 silver rings. Oppenheimer inserted 1500 rings and did not find any complication such as inflammation, fever, or infection as a result of the ring insertion. In regard to the Lippes loop and other polyethylene devices, investigators in 1 study accumulated 15,419 woman-months experience with 1531 first insertions. The incidence of PID was 1.6%. Another group of investigators reported a 7.7% incidence of PID in a group of 623 clinic patients who were using IUDs. Lippes reported a rate of 0.6/100 woman-years for 1673 patients during 16,077 woman-months of exposure. It has been suggested that the IUD tails may be responsible for the passage of vaginal bacteria into the uterus. A lower incidence of bacterial contamination of the uterine cavity with copper devices as compared with others has been demonstrated. It has been observed that the PID rate in 750 women using the Copper T-200 for 1 year after insertion was 2.1%. A group of investigators reviewed the incidence of PID in 516 never pregnant women using Copper 7 IUD for 9080 woman-months at 4 centers. The incidence of PID varied between 0.9-1.4% per year in different centers. It was found that the rate of PID in women using Progestaserts was between 2 and 3.1% per year, and it was strongly correlated with the history of PID. Comparative trials are reported along with studies relating to the use of IUDs and an increased risk of ectopic pregnancy. A positive relationship between the duration of IUD use the risk of ectopic pregnancy has been observed. An investigator reviewed his experiences of 711 ectopic pregnancies; 70 of these were associated with the use of an IUD, which comprised 10% of all the ectopics in a 9-year period. A prevalence of about 21% extrauterine pregnancies among the Progestasert IUD users, as against 2.5% among other IUD users. Little information is available regarding whether, after removal of the IUD, the woman is still at a higher risk of tubal pregnancy at a subsequent pregnancy.^ieng

The role of endometrial alkaline phosphatase in the mechanism of action of the intrauterine device in the rat.

PIP: The role of endometrial alkaline phosphatase in the mechanism of action of the IUD was investigated in the rat. A silk thread suture was inserted into the lumen of 1 horn of the rat uterus. Implantation sites were observed on Day 10 of pregnancy. Endometrial acid and alkaline phosphatase levels were measured in some of the animals on Day 5 of pregnancy. The suture significantly raised the endometrial alkaline (p .001) and acid (p .005) phosphatase levels in the horn with the IUD. When the IUD was removed on Day 2 of pregnancy a significant fall (p .001) in alkaline phosphatase was observed on Day 5 of pregnancy. 8 of 12 rats from whom the IUD had been removed on Day 2 showed evidence of implantation. It is suggested that the suture may be exerting its contraceptive effect by preventing implantation by raising endometrial alkaline phosphatase levels.^ieng

Effect of intrauterine administration of antiprostaglandin drugs on implantation in the rat.

Three antiprostaglandin drugs, acetylsalicylic acid, indomethacin and ibuprofen, were administered intraluminally at different does to pregnant rats on Day 4 of pregnancy. It was observed that ibuprofen administered at a dose of 400 micrograms inhibited implantation in all rats. Ibuprofen when administered at a lower dose of 100 or 200 micrograms on Day 4 of pregnancy or at a dose of 400 micrograms on Day 3 or 5 of pregnancy did not exert such an antiimplantation effect. Acetylsalicylic acid and indomethacin also induced, but to a lesser extent, an antiimplantation effect when administered at a dose of 400 micrograms on Day 4 of pregnancy.

A comparative controlled trial in rural Thailand of three intrauterine devices.

PIP: The 1st controlled clinical IUD trial carried out as a rural field trial in a developing country was conducted in Thailand. 2452 insertions of 3 different IUDs -- Lippes Loop C, Antigon-F, and Ypsilon -- were made and followed up for a 2-year period. The women were fully informed volunteers who were screened for contraindications to IUDs. The 3 groups were comparable in age, parity, open birth intervals, previous contraceptive use, and hematocrit level. Life table analysis indicated comparable cumulative pregnancy rates for the 3 devices at 12 and at 24 months. Antigon-F had significantly lower expulsion rates and Lippes C the highest expulsion rates of the 3. In fact, expulsion rates for this entire study were atypically high when compared to other studies. This may be attributable to differences in subject characteristics, ethnic factors, or follow-up procedures. The cumulative removal rates for all reasons were found to be comparable for the 3 devices. During most of the period of observation, cumulative total termination rates for all events were significantly lower for the Antigon-F, which recommends this as the preferable IUD for this population.^ieng

The antifertility effect of 2', 3', 5'tri-O-acetyl-6-azauridine. I.

PIP: In this investigation, the effect of orally administered TA-AZUR, 2',3',5'tri-0-acetyl-6-azauridine) on pregnancy was studied after administration of the compound to mature female rats, at various doses and on different days of pregnancy. TA-AZUR was administered at doses of 200, 300, and 400 mg/kg to 20, 15, and 10 rats respectively. There was an inhibition of implantation in 60% of rats which had received a dose of 200 mg/kg and 80% inhibition in rats on 300 and 400 mg/kg. It was also shown that at 200 mg/kg, TA-AZUR did not have any antifertility activity when administered on Day 1 or 2 of pregnancy. It appeared to possess a greater effect when administered later in pregnancy, the most significant antifertility activity being shown when administered on Days 6, 7, 8, 9, and 10 of pregnancy. The compound showed no evidence of toxicity at reported doses. The mode of action seems to be more as an abortifacient one.^ieng

Antifertility screening of plants. 3. Effect of six indigenous plants on early pregnancy in albino rats.

PIP: The effect of 6 indigenous plants on early pregnancy in albino rats was tested by a screening procedure standardized in this laboratory. Pe troleum ether, alcoholic, and aqueous extracts of each plant were tested for antifertilizing, antizygotic, blastocystotoxic, antiimplantation, and early abortifacient activity. The aqueous extract of Ocimum sanctum Linn. leaves and alcoholic extract of Polygonum hydropiper Linn. roots showed encouraging results while the extracts of Abroma augusta Linn. roots, Calotropis gigantea Linn. flowers and leaves, Michaelia champaka Linn. unripe fruit, and Plumbago rosea Linn. roots did not show any antiimplantation activity. None of the rats delivered to experimental rats showed evidence of teratogenicity up to the age of 1 month.^ieng

Antifertility screening of plants. VI. Effect of five indigenous plants on early pregnancy in albino rats.

PIP: The petroleum ether, alcoholic and aqueous extracts of Apium graveolens Linn., Butea monosperma Lam. Kuntz., and Gossypium herbaceum Linn., the aqueous extract of Aloe Barbadensis Mill.Syn., and the juice of unripe fruits of Ananas comosus were tested on albino rats by a method which detects any antizygotic, blastocystotoxic, antiimplantation, and early abortifacient activity. The extracts were administered for 1-7 days. The dosages for A. graveolens, B. monosperma, and G. herbaceum were 100 mg/kg. 50 ml of A. comosus juice was administered daily. Dosages of 100, 200, and 500 mg/kg of A. barbadensis were given. With the exception of A. comosus, none of the plants showed any antiimplantation activity. The juice of the unripe fruits of A. comosus demonstrated encouraging antiimplantation activity showing 40% of implants only.^ieng

The quest for a herbal contraceptive.

PIP: An oral herbal contraceptive would allow couples control their fertility without consulting a health worker, which in turn would likely markedly increase the number of couples practicing family planning. Other advantages of such a contraceptive would include the familiarity rural people have with herbal medicines, the fewer side effects associated with herbal preparations, their ready availability from local sources, and protection of privacy. There are many references to plants in India with antifertility properties. Since 1966, the Indian Council of Medical Research (ICMR) has been conducting research to identify a herbal contraceptive, as have other organizations. Plants that have exhibited antifertility activity in clinical trials include Hibiscus rosasinensis (benzene extract of the flower petals suppresses implantation); Rudrapushpaka (extract of the flower petals prevents pregnancy); Embelia ribes (pregnancy prevention); Davcus carota, Butea monosperma, and Sapindus trifoliatis (seeds have an anti-implantation effect); and Mentha arvensis (leaves have anti-implantation effect). The Central Drug Research Institute in Lucknow, India, in collaboration with the US National Institutes of Health, the World Health Organization, and the ICMR confirm anti-implantation activity in Ferula jaeschkeana, Bupleurum marginatum, Lepidium capitatum, Caesalpinia sepiaria, Lonicera japonica, Juniperus communis, Lotus corniculatus, Lamium allum, and Acacia farnesiana. In China, scientists have evaluated the cotton-seed extract gossypol as a male contraceptive. They are now studying the possible antifertility effect on men of the plant Tripterygium wilfordii. From all the aforementioned plants as well as others under investigation, three possible types of contraceptives could be developed: an anti-ovulatory contraceptive; a postcoital contraceptive; and a male contraceptive. Some obstacles to their development include difficulties in obtaining adequate quantities of the herbs, a shortage of clinical pharmacologists and clinicians interested in conducting clinical trials, and lack of long-term financial support.^ieng