Agrobacterium mediated transformation of banana (Musa sp.) cv. Sukali Ndiizi (ABB) with a modified Carica papaya cystatin (CpCYS) gene

Conventional banana breeding for pest and disease resistance is a very difficult and slow process due to the limited sources of resistance, sterility of cultivated banana varieties, high polyploidy levels, long cropping cycle and the lack of rapid screening methods. Molecular breeding using the transgenic approach with candidate genes such as cystatins offers an alternative method to banana improvement. Cystatin proteins inhibit the activity of cysteine proteases responsible for the breakdown of dietary proteins in the gut of many pests including nematodes resulting in protein deficiency. In this study, the papaya cystatin gene was introduced into the banana genome. Embryogenic cell suspension (ECS) cultures of the banana cultivar Sukali Ndiizi (ABB) were used as explants material for the successful transformation of banana. The Carica papaya cystatin gene (CpCYS-Mut89) previously modified to improve its inhibitory potential against banana pests was introduced into this cultivar using Agrobacterium tumefaciens, strain LBA4404 and the gus reporter gene was used to observe successful transformation process. We report the successful protocol for routine transformation of this cultivar, which was completed in six months with plant regeneration observed at a frequency of 23%. An additional four months was required to multiply the regenerant lines in order to have at least 20 plants per line for downstream challenging studies. Putatively transgenic plants were analyzed by PCR using hpt and CpCYS-Mut89 specific primers to confirm the presence of transgenes. Out of 28 selected lines, 27 were positive for both hpt and CpCYS-Mut89 transgenes giving 96.4% transformation efficiency. Five lines were then selected on the basis of putative PCR positives and a Southern blot analysis gave hybridization signals with 1 to 4 copy number integration patterns characteristic of Agrobacterium mediated transformation. These results confirm stable gene integration in East African banana cultivar cv. Sukali Ndiizi (genome group ABB) through an efficient Agrobacterium-mediated transformation protocol described for routine use in future improvement of this crop with genes of economic importance.Keywords: Cystatins, banana, Agrobacterium mediated transformation, southern blotAfrican Journal of Biotechnology Vol. 12(15), pp. 1811-181

Agrobacterium mediated transformation of banana (Musa sp.) cv. Sukali Ndiizi (ABB) with a modified Carica papaya cystatin (CpCYS) gene

Conventional banana breeding for pest and disease resistance is a very difficult and slow process due to the limited sources of resistance, sterility of cultivated banana varieties, high polyploidy levels, long cropping cycle and the lack of rapid screening methods.Molecular breeding using the transgenic approach with candidate genes such as cystatins offers an alternative method to banana improvement. Cystatin proteins inhibit the activity of cysteine proteases responsible for the breakdown of dietary proteins in the gut of many pests including nematodes resulting in protein deficiency. In this study, the papaya cystatin gene was introduced into the banana genome. Embryogenic cell suspension (ECS) cultures of the banana cultivar Sukali Ndiizi (ABB) were used as explants material for the successful transformation of banana. The Carica papaya cystatin gene (CpCYS-Mut89) previously modified to improve its inhibitory potential against banana pests was introduced into this cultivar using Agrobacterium tumefaciens, strain LBA4404 and the gus reporter gene was used to observe successful transformation process. We report the successful protocol for routine transformation of this cultivar, which was completed in six months with plant regeneration observed at a frequency of 23%. An additional four months was required to multiply the regenerant lines in order to have at least 20 plants per line for downstream challenging studies. Putatively transgenic plants were analyzed by PCR using hpt and CpCYS - Mut89 specific primers to confirm the presence of trans genes. Out of 28 selected lines, 27 were positive for both hpt and CpCYS - Mut89 transgenes giving 96.4 % transformation efficiency. Fivelines were then selected on the basis of putative PCR positives and a Southern b lot analysis gave hybridization signals with 1 to 4 copy number integration patterns characteristic of Agrobacterium mediated transformation. These results confirm stable gene integration in East African banana cultivar cv. Sukali Ndiizi (genome group ABB) through an efficient Agrobacterium - mediated transformation protocol described for routine use in future improvement of this crop with genes of economic importance

Agrobacterium mediated transformation of banana (Musa sp.) cv. Sukali Ndiizi (ABB) with a modified Carica papaya cystatin (CpCYS) gene

Conventional banana breeding for pest and disease resistance is a very difficult and slow process due to the limited sources of resistance, sterility of cultivated banana varieties, high polyploidy levels, long cropping cycle and the lack of rapid screening methods. Molecular breeding using the transgenic approach with candidate genes such as cystatins offers an alternative method to banana improvement. Cystatin proteins inhibit the activity of cysteine proteases responsible for the breakdown of dietary proteins in the gut of many pests including nematodes resulting in protein deficiency. In this study, the papaya cystatin gene was introduced into the banana genome. Embryogenic cell suspension (ECS) cultures of the banana cultivar Sukali Ndiizi (ABB) were used as explants material for the successful transformation of banana. The Carica papaya cystatin gene (CpCYS-Mut89) previously modified to improve its inhibitory potential against banana pests was introduced into this cultivar using Agrobacterium tumefaciens, strain LBA4404 and the gus reporter gene was used to observe successful transformation process. We report the successful protocol for routine transformation of this cultivar, which was completed in six months with plant regeneration observed at a frequency of 23%. An additional four months was required to multiply the regenerant lines in order to have at least 20 plants per line for downstream challenging studies. Putatively transgenic plants were analyzed by PCR using hpt and CpCYS-Mut89 specific primers to confirm the presence of transgenes. Out of 28 selected lines, 27 were positive for both hpt and CpCYS-Mut89 transgenes giving 96.4% transformation efficiency. Five lines were then selected on the basis of putative PCR positives and a Southern blot analysis gave hybridization signals with 1 to 4 copy number integration patterns characteristic of Agrobacterium mediated transformation. These results confirm stable gene integration in East African banana cultivar cv. Sukali Ndiizi (genome group ABB) through an efficient Agrobacterium-mediated transformation protocol described for routine use in future improvement of this crop with genes of economic importance.Bioversity International through the National Banana Research Programmme of the National Agricultural Research Organization, Uganda.http://www.academicjournals.org/AJBam201

Young HIV-Infected Children and Their Adult Caregivers Prefer Tablets to Syrup Antiretroviral Medications in Africa

Background: Provision of anti-retroviral therapy (ART) for HIV-infected children is complicated using syrup formulations, which are costlier than tablets, harder to transport and store and difficult for health-workers to prescribe and caregivers to administer. Dispersible/crushable tablets may be more appropriate. We studied the acceptability of syrups and scored tablets among young children who used both in the AntiRetroviral Research fOr Watoto (ARROW) trial. Methods: ARROW is an ongoing randomized trial of paediatric ART monitoring and treatment strategies in 1206 children in Uganda and Zimbabwe. 405 children initially received syrups of combination ART including Nevirapine, Zidovudine, Abacavir and Lamivudine before changing, when reaching the 12-,15 kg weightband, to scored adult-dose tablets prescribed according to WHO weightband tables. Caregiver expectations and experiences were collected in questionnaires at their last visit on syrups and after 8 and 24 weeks on tablets. Results: Questionnaires were completed by caregivers of 267 children (median age 2.9 years (IQR 2.5, 3.4)). At last visit on syrups, 79 % caregivers reported problems with syrups, mostly related to number, weight, transportation and conspicuousness of bottles. Difficulties taking tablets were expected by 127(48%) caregivers; however, after 8 and 24 weeks, only 26 % and 18 % reported their children had problems with tablets and no problems were reported with transportation/conspicuousness. Taste, swallowing or vomiting were reported as problems ‘sometimes/often ’ for 14%, 9%

Prevalence and factors associated with traditional herbal medicine use among patients on highly active antiretroviral therapy in Uganda

<p>Abstract</p> <p>Background</p> <p>In Africa, herbal medicines are often used as primary treatment for Human immunodeficiency virus (HIV) related problems. Concurrent use of traditional herbal medicines (THM) with antiretroviral drugs (ARVs) is widespread among HIV infected patients. However, the extent of THM use is not known in most settings in Sub-Saharan Africa. This study aimed at determining the prevalence and factors associated with THM use among HIV infected patients on highly active antiretroviral therapy (HAART) attending The AIDS Support Organization (TASO) in Uganda. TASO is a non-governmental organization devoted to offering HIV/AIDS care and treatment services in the population.</p> <p>Methods</p> <p>This was a cross-sectional study carried out in two TASO treatment centres in Uganda among 401 randomly selected eligible participants. We included participants who were 18 years and above, were enrolled on HAART, and consented to participate in the study. Data was collected using an interviewer-administered semi-structured questionnaire. THM use referred to someone who had ever used or was currently using herbal medicine while on highly active antiretroviral therapy (HAART) by the time of the study. Data was captured in Epi-data version 3.1 and exported to STATA version 9.0 for analysis.</p> <p>Results</p> <p>The prevalence of THM use was 33.7%. Patients on HAART for < 4 years were more likely to use THM (OR = 5.98, 95% CI 1.13 - 31.73) as well as those who experienced HAART side effects (OR = 3.66, 95% CI: 1.15 - 11.68). Older patients (≥39 years) were less likely to use THM (OR = 0.26 95% CI: 0.08 - 0.83). Participants with HAART adherence levels > 95% were less likely to use THM (OR = 0.09, 95% CI 0.01 - 0.65).</p> <p>Conclusion</p> <p>The prevalence of THM use among participants on HAART was high. This raises clinical and pharmacological concerns that need attention by the health care service providers.</p

Neuropsychiatric manifestations and sleep disturbances with dolutegravir-based antiretroviral therapy versus standard of care in children and adolescents: a secondary analysis of the ODYSSEY trial

BACKGROUND: Cohort studies in adults with HIV showed that dolutegravir was associated with neuropsychiatric adverse events and sleep problems, yet data are scarce in children and adolescents. We aimed to evaluate neuropsychiatric manifestations in children and adolescents treated with dolutegravir-based treatment versus alternative antiretroviral therapy. METHODS: This is a secondary analysis of ODYSSEY, an open-label, multicentre, randomised, non-inferiority trial, in which adolescents and children initiating first-line or second-line antiretroviral therapy were randomly assigned 1:1 to dolutegravir-based treatment or standard-of-care treatment. We assessed neuropsychiatric adverse events (reported by clinicians) and responses to the mood and sleep questionnaires (reported by the participant or their carer) in both groups. We compared the proportions of patients with neuropsychiatric adverse events (neurological, psychiatric, and total), time to first neuropsychiatric adverse event, and participant-reported responses to questionnaires capturing issues with mood, suicidal thoughts, and sleep problems. FINDINGS: Between Sept 20, 2016, and June 22, 2018, 707 participants were enrolled, of whom 345 (49%) were female and 362 (51%) were male, and 623 (88%) were Black-African. Of 707 participants, 350 (50%) were randomly assigned to dolutegravir-based antiretroviral therapy and 357 (50%) to non-dolutegravir-based standard-of-care. 311 (44%) of 707 participants started first-line antiretroviral therapy (ODYSSEY-A; 145 [92%] of 157 participants had efavirenz-based therapy in the standard-of-care group), and 396 (56%) of 707 started second-line therapy (ODYSSEY-B; 195 [98%] of 200 had protease inhibitor-based therapy in the standard-of-care group). During follow-up (median 142 weeks, IQR 124–159), 23 participants had 31 neuropsychiatric adverse events (15 in the dolutegravir group and eight in the standard-of-care group; difference in proportion of participants with ≥1 event p=0·13). 11 participants had one or more neurological events (six and five; p=0·74) and 14 participants had one or more psychiatric events (ten and four; p=0·097). Among 14 participants with psychiatric events, eight participants in the dolutegravir group and four in standard-of-care group had suicidal ideation or behaviour. More participants in the dolutegravir group than the standard-of-care group reported symptoms of self-harm (eight vs one; p=0·025), life not worth living (17 vs five; p=0·0091), or suicidal thoughts (13 vs none; p=0·0006) at one or more follow-up visits. Most reports were transient. There were no differences by treatment group in low mood or feeling sad, problems concentrating, feeling worried or feeling angry or aggressive, sleep problems, or sleep quality. INTERPRETATION: The numbers of neuropsychiatric adverse events and reported neuropsychiatric symptoms were low. However, numerically more participants had psychiatric events and reported suicidality ideation in the dolutegravir group than the standard-of-care group. These differences should be interpreted with caution in an open-label trial. Clinicians and policy makers should consider including suicidality screening of children or adolescents receiving dolutegravir

Dolutegravir twice-daily dosing in children with HIV-associated tuberculosis: a pharmacokinetic and safety study within the open-label, multicentre, randomised, non-inferiority ODYSSEY trial

Background: Children with HIV-associated tuberculosis (TB) have few antiretroviral therapy (ART) options. We aimed to evaluate the safety and pharmacokinetics of dolutegravir twice-daily dosing in children receiving rifampicin for HIV-associated TB. Methods: We nested a two-period, fixed-order pharmacokinetic substudy within the open-label, multicentre, randomised, controlled, non-inferiority ODYSSEY trial at research centres in South Africa, Uganda, and Zimbabwe. Children (aged 4 weeks to <18 years) with HIV-associated TB who were receiving rifampicin and twice-daily dolutegravir were eligible for inclusion. We did a 12-h pharmacokinetic profile on rifampicin and twice-daily dolutegravir and a 24-h profile on once-daily dolutegravir. Geometric mean ratios for trough plasma concentration (Ctrough), area under the plasma concentration time curve from 0 h to 24 h after dosing (AUC0–24 h), and maximum plasma concentration (Cmax) were used to compare dolutegravir concentrations between substudy days. We assessed rifampicin Cmax on the first substudy day. All children within ODYSSEY with HIV-associated TB who received rifampicin and twice-daily dolutegravir were included in the safety analysis. We described adverse events reported from starting twice-daily dolutegravir to 30 days after returning to once-daily dolutegravir. This trial is registered with ClinicalTrials.gov (NCT02259127), EudraCT (2014–002632-14), and the ISRCTN registry (ISRCTN91737921). Findings: Between Sept 20, 2016, and June 28, 2021, 37 children with HIV-associated TB (median age 11·9 years [range 0·4–17·6], 19 [51%] were female and 18 [49%] were male, 36 [97%] in Africa and one [3%] in Thailand) received rifampicin with twice-daily dolutegravir and were included in the safety analysis. 20 (54%) of 37 children enrolled in the pharmacokinetic substudy, 14 of whom contributed at least one evaluable pharmacokinetic curve for dolutegravir, including 12 who had within-participant comparisons. Geometric mean ratios for rifampicin and twice-daily dolutegravir versus once-daily dolutegravir were 1·51 (90% CI 1·08–2·11) for Ctrough, 1·23 (0·99–1·53) for AUC0–24 h, and 0·94 (0·76–1·16) for Cmax. Individual dolutegravir Ctrough concentrations were higher than the 90% effective concentration (ie, 0·32 mg/L) in all children receiving rifampicin and twice-daily dolutegravir. Of 18 children with evaluable rifampicin concentrations, 15 (83%) had a Cmax of less than the optimal target concentration of 8 mg/L. Rifampicin geometric mean Cmax was 5·1 mg/L (coefficient of variation 71%). During a median follow-up of 31 weeks (IQR 30–40), 15 grade 3 or higher adverse events occurred among 11 (30%) of 37 children, ten serious adverse events occurred among eight (22%) children, including two deaths (one tuberculosis-related death, one death due to traumatic injury); no adverse events, including deaths, were considered related to dolutegravir. Interpretation: Twice-daily dolutegravir was shown to be safe and sufficient to overcome the rifampicin enzyme-inducing effect in children, and could provide a practical ART option for children with HIV-associated TB

The pharmacokinetics and acceptability of lopinavir/ritonavir minitab sprinkles, tablets, and syrups in african HIV-infected children.

BACKGROUND: Guidelines recommend lopinavir/ritonavir (LPV/r) as first- and second-line therapy for young and older HIV-infected children, respectively. Available formulations have limitations making their widespread use complex. METHODS: An open-label comparative bioavailability (randomized crossover) study compared a novel twice-daily minitab sprinkle formulation (40 mg/10 mg, Cipla Pharmaceuticals) versus innovator syrup in HIV-infected Ugandan infants aged 3 to &lt;12 months (cohort A) and children aged 1-4 years (cohort B) and versus Cipla tablets (100/25 mg) in children aged 4 to &lt;13 years (cohort C). Twelve-hour intensive pharmacokinetic sampling after observed LPV/r intake (plus 2 nucleoside reverse transcriptase inhibitors) following World Health Organization 2010 dosing with food was performed 4 weeks after enrollment. Children then switched formulation; sampling was repeated at week 8. Acceptability data were also collected. RESULTS: Seventy-seven infants/children were included in cohort A (n = 19)/B (n = 26)/C (n = 32). Among 132 evaluable pharmacokinetic profiles, there were 13/21/25 within-child comparisons in cohort A/B/C. For minitabs versus syrup, geometric mean [95% confidence interval (CI)] AUC0-12h was 88.6 (66.7-117.6) versus 77.6 (49.5-121.5) h·mg/L in cohort A [geometric mean ratio (GMR) (90% CI) = 1.14 (0.71 to 1.85)] and 138.7 (118.2 to 162.6) versus 109.1 (93.7 to 127.1) h·mg/L in cohort B [GMR (90% CI) = 1.27 (1.10 to 1.46)]. For minitabs versus tablets, geometric mean (95% CI) AUC0-12h was 83.1 (66.7 to 103.5) versus 115.6 (103.0 to 129.7) h·mg/L; GMR (90% CI) = 0.72 (0.60 to 0.86). Subtherapeutic levels (&lt;1.0 mg/L) occurred in 0 (0%)/2 (15%) minitabs/syrup in infants (P = 0.48), no children aged 1-4 years and 4 (16%)/1 (4%) minitabs/tablets (P = 0.35). About 13/17 (76%) and 19/26 (73%) caregivers of infants and children aged 1-4 years, respectively, chose to continue minitabs after week 8, mainly for convenience; only 7/29 (24%) older children (five &lt;6 years) remained on minitabs. CONCLUSIONS: LPV/r exposure from minitabs was comparable with syrup, but lower than tablets, with no significant differences in subtherapeutic concentrations. Minitabs were more acceptable than syrups for younger children, but older children preferred tablets