Identification of bottlenecks in the accumulation of cyclic fatty acids in camelina seed oil.

Modified fatty acids (mFA) have diverse uses; for example, cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics and cosmetics. The expression of mFA-producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural-occurring source plants. Thus, to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co-expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA-accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon co-expression of SfLPAT with EcCPS, di-CPA-PC increased by ~50% relative to lines expressing EcCPS alone with the di-CPA-PC primarily observed in the embryonic axis and mono-CPA-PC primarily in cotyledon tissue. EcCPS-SfLPAT lines revealed a redistribution of CPA from the sn-1 to sn-2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. The identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops.

Coexpressing Escherichia coli cyclopropane synthase with Sterculia foetida Lysophosphatidic acid acyltransferase enhances cyclopropane fatty acid accumulation.

Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation.

Is withdrawal of nocturnal hyperhydration possible in children with primary hyperoxaluria treated with RNAi?

Primary hyperoxaluria type 1 is a rare genetic disorder caused by bi-allelic pathogenic variants in the AGXT gene leading to an overproduction of oxalate which accumulates in the kidneys in the form of calcium oxalate crystals. Thus, patients may present with recurrent nephrocalcinosis and lithiasis, with progressive impairment of the  renal function and eventually kidney failure.  There is no specific treatment besides liver-kidney transplantation, and pre-transplantation management by 24 h-hyperhydration, crystallisation inhibitors and high-dose pyridoxine has a high negative impact on quality of life, especially because of the discomfort due to nocturnal hyperhydration. Since 2020, lumasiran, an RNA-interfering therapy, has been approved for the treatment of primary hyperoxaluria type 1 in adults and children. However, to date, there are no recommendations regarding the discontinuation of other supportive measures during RNAi therapy. In this report, we present two patients with primary hyperoxaluria type 1 who were treated with lumasiran and stopped nocturnal hyperhydration with positive outcomes, i.e. normal urinary oxalate, absence of crystalluria, stable kidney function and improved well-being. These data suggest that discontinuing nocturnal hydration may be safe in children responding to lumasiran, and may have a positive impact on their quality of life. Additional data are needed to update treatment recommendations.

Survival of Tunneled Double Lumen-Cuffed Catheters in Children on Maintenance Hemodialysis - A Retrospective Cohort Study.

Introduction: Survival of tunneled cuffed catheters (TCC), used widely in children, is complicated by infections and catheter dysfunction. In resource limited settings, where risk of complications could be higher and waiting period for transplantation longer, catheter survival determines patient survival. This study was conducted to determine infection free catheter survival rates, incidence of catheter failure and associated risk factors. Methods: Children <18 years of age receiving maintenance hemodialysis through TCC at nephrology division of a pediatric hospital, over a period of 6 years. Data was collected with consecutive selection by a complete enumeration technique from pre-collected data sheets in the records. Exposure detected were catheter infections, thrombosis, and mechanical complications. Results: Forty-five TCCs in 36 children studied for 12,590 catheter days showed catheter failure in 36%, due to catheter related infections in 75% and mechanical complications in 25%. The incidence of complications per 1000 catheter days was 1.19 infection, 1.03 thrombus, and 0.39 mechanical. Catheter-related blood stream infection (CRBSI) (15/36) was associated with thrombus in nine and led to mortality in three. The mean infection free catheter survival was 449 ± 42 days for cohort with 388 ± 38 days in Group A (premature catheter removal) and 593 ± 43 days in Group B (elective removal) (P = 0.03). Catheterization duration of 267 days predicted CRBSI (sensitivity 93%, specificity 66.7%) with area under the curve of 0.808. Conclusions: Median infection free catheter survival was 449 days with catheter failure in 36%. CRBSI was the main cause of failure. Duration of catheterization greater than 267 days was a predictor of CRBSI.

Immuno-profiling of Brucella proteins for developing improved vaccines and DIVA capable serodiagnostic assays for brucellosis.

Brucellosis remains a worldwide zoonotic disease with a serious impact on public health and livestock productivity. Controlling brucellosis in livestock is crucial for limiting human infections in the absence of effective human vaccines. Brucellosis control measures are majorly dependent on rigorous monitoring of disease outbreaks and mass vaccination of livestock. Live attenuated vaccines are available for livestock vaccination that play a vital role in brucellosis control programs in many countries. Even though the existing animal vaccines confer protection against brucellosis, they carry some drawbacks, including their infectivity to humans and interference with sero-monitoring. The available serodiagnostic assays for brucellosis depend on detecting anti-LPS antibodies in the serum. Since diagnosis plays a vital role in controlling brucellosis, developing improved serodiagnostic assays with enhanced specificity, sensitivity and DIVA capability is required. Therefore, it is essential to identify novel antigens for developing improved vaccines and serodiagnostic assays for brucellosis. In the present study, we performed a high throughput immunoprofiling of B. melitensis protein microarray using brucellosis-positive human and animal serum samples. The screening identified several serodominant proteins of Brucella that exhibited common or differential reactivity with sera from animals and humans. Subsequently, we cloned, expressed, and purified ten serodominant proteins, followed by analyzing their potential to develop next-generation vaccines and improved serodiagnostic assays for brucellosis. Further, we demonstrated the protective efficacy of one of the serodominant proteins against the B. melitensis challenge in mice. We found that the seroreactive protein, Dps (BMEI1980), strongly reacted with brucellosis-positive serum samples, but it did not react with sera from B. abortus S19-vaccinated cattle, indicating DIVA capability. A prototype lateral flow assay and indirect ELISA based on Dps protein exhibited high sensitivity, specificity, and DIVA capability. Thus, the present study identified promising candidates for developing improved vaccines and affordable, DIVA-capable serodiagnostic assays for animal and human brucellosis.

Prevalence and subtypes of hypertension in normal-weight and obese Indian adolescents: a cross-sectional study.

This cross-sectional study examined the prevalence, stages, subtypes of hypertension, and the associated risk factors in adolescent school children in Western India. We screened 2,644 adolescents, from 10 different private and government schools in urban and rural areas for hypertension, as defined by the 2017 Clinical Practice Guidelines. The association of stages and subtypes with age, gender, body mass index, type of school, and place of residence was analysed. 197 children (7.5%) had hypertension; 170 (6.4%) had stage I, 27 (1%) had stage II and 76 (2.9%) had elevated blood pressure (EBP). The risk of EBP was higher in children > 15 years of age (p = 0.006). Compared with normal-weight children, obese, and overweight children had a higher risk of hypertension [odds ratio (OR) 9 (5.84, 13.88) and 3.77 (2.59, 5.48) respectively], whereas underweight children had a lower risk [OR 0.39 (0.16, 0.98)]. Normal-weight hypertension was seen in 5.2% and was higher in children from government schools (9.4%). Systolic-diastolic hypertension (SDH) was the most common subtype, seen in 136 (5.1%). SDH was more common in girls, in rural children, and in those with stage II hypertension. Isolated diastolic hypertension, seen in 51 (1.9%), was more common in boys, in urban children, and in those with EBP.

Clinico-pathological Profile and Outcome of C-3 Glomerulopathy in Indian Children.

INTRODUCTION: There is paucity of data of C3 glomerulopathy in Indian children. METHODS: First Indian pediatric case series where consecutive renal biopsies done over a period of ten years were reviewed to identify those patients who had isolated or predominant C3 deposits on immunofluorescent microscopy, fulfilling the criteria for C-3 glomerulopathy. The clinical, biochemical, serological, histopathological profile, eGFR and the need for renal replacement therapy was analyzed. RESULTS: Eighteen patients, comprising 5.3% (18/298) of all renal biopsies, had C3 glomerulopathy, four with Dense Deposit Disease (DDD) and fourteen with C3 Glomerulonephritis (C3GN) with a median follow-up of 38.2 months. Median age of presentation was 7.45±3.03 years (2.5yrs- 13.5yrs) with nine boys and nine girls. Presentation was nephrotic syndrome in seven (39%), acute nephritic syndrome in three (16.7%), hematuria in five (27.7%) and acute kidney injury in three (16.7%). Median eGFR was 69 ml/min/1.73m2 (8.2-107 ml/min/1.73m2). Hematuria was seen in 16 (88%), proteinuria in 18 (100%) and low C3 in 16 (88%) at the time of presentation. Mesangioproliferative glomerulonephritis was the predominant pattern in DDD while C3GN showed a mix of mesangioproliferative, membranoproliferative, endocapillary and crescentic GN (p = 0.43).Complete or partial remission was seen in seven patients who received long term alternate day steroids alone or with added mycophenolate mofetil. The cumulative patient survival was 70.8%. Kaplan Meir analyses for renal survival without progression to ESRD was 60.2% at one year and 48.1% at five and ten years. CONCLUSION: Interstitial fibrosis and tubular atrophy on renal biopsy was an independent predictor of adverse renal outcome in the cohort (p = 0.013, HR8.1;95% CI -1.6-42).

[Hydroxy(tosyloxy)iodo]benzene mediated alpha-azidation of ketones.

Reaction of various ketones with [hydroxy(tosyloxy)iodo]benzene (HTIB) followed by treatment of the alpha-tosyloxy ketones thus generated in situ with NaN3 offers a one-pot procedure for the synthesis of alpha-azido ketones. The HTIB used in this conversion may also be generated in situ by using iodosobenzene in combination with p-toluene-sulphonic acid.