Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.

A carboxysome-based CO2 concentrating mechanism for C3 crop chloroplasts: advances and the road ahead.

The introduction of the carboxysome-based CO2 concentrating mechanism (CCM) into crop plants has been modelled to significantly increase crop yields. This projection serves as motivation for pursuing this strategy to contribute to global food security. The successful implementation of this engineering challenge is reliant upon the transfer of a microcompartment that encapsulates cyanobacterial Rubisco, known as the carboxysome, alongside active bicarbonate transporters. To date, significant progress has been achieved with respect to understanding various aspects of the cyanobacterial CCM, and more recently, different components of the carboxysome have been successfully introduced into plant chloroplasts. In this Perspective piece, we summarise recent findings and offer new research avenues that will accelerate research in this field to ultimately and successfully introduce the carboxysome into crop plants for increased crop yields.

Plant-based carboxysomes: another step toward increased crop yields.

Synthetically reconstructed carboxysomes form the basis of CO2-concentrating mechanisms (CCMs) that could enhance the photosynthetic efficiency of crops and improve yield. Recently, Chen et al. revealed another step toward the reconstruction of bacterial carboxysomes in plants, reporting the formation of almost-complete carboxysomes in the chloroplast of Nicotiana tabacum.

Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts.

A long-term strategy to enhance global crop photosynthesis and yield involves the introduction of cyanobacterial CO2-concentrating mechanisms (CCMs) into plant chloroplasts. Cyanobacterial CCMs enable relatively rapid CO2 fixation by elevating intracellular inorganic carbon as bicarbonate, then concentrating it as CO2 around the enzyme Rubisco in specialized protein micro-compartments called carboxysomes. To date, chloroplastic expression of carboxysomes has been elusive, requiring coordinated expression of almost a dozen proteins. Here we successfully produce simplified carboxysomes, isometric with those of the source organism Cyanobium, within tobacco chloroplasts. We replace the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco large and small subunit genes, along with genes for two key α-carboxysome structural proteins. This minimal gene set produces carboxysomes, which encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO2. This result demonstrates the formation of α-carboxysomes from a reduced gene set, informing the step-wise construction of fully functional α-carboxysomes in chloroplasts.

Assessment of Clinical Practices for Crushing Medication in Geriatric Units.

OBJECTIVES: To assess the modification of the form of medication and evaluate staff observance of good clinical practices. DESIGN: One-day assessment of clinical practices. SETTING: 17 geriatrics units in the 3 Teaching Hospitals of Paris-Sud (APHP), France. PARTICIPANTS: Elderly in-patients with difficulties swallowing capsules and tablets. MEASUREMENTS: Assessment of target-patient prescriptions and direct observation of nurses' medical rounds. RESULTS: 155/526 in-patients (29.5%) were unable to swallow tablets or capsules: 98 (40.3%) in long-term care, 46 patients (23.8%) in the rehabilitation unit and 11 (12.2%) in the acute care unit (p = .005). In thirty-nine (27.3%) of the 143 prescriptions studied all tablets were safe to crush and all capsules were safe to open. In 104 cases, at least one medication could not be safely modified, including 26 cases (18.2%) in which none of the prescribed drugs were safe to crush or open. In 48.2% of the 110 medications that were crushed, crushing was forbidden, and presented a potential threat in 12.7% of cases or a reduced efficacy in 8.2% of cases. Crushing methods were rarely appropriate: no specific protective equipment was used (81.8%), crushing equipment was shared between patients without cleaning (95.1%), medications were spilled or lost (69.9%). The method of administration was appropriate (water, jellified water) in 25% of the cases, questionable (soup, coffee, compote, juice, cream) in 55% of the cases and unacceptable (laxative) in 21% of the cases. CONCLUSION: Management of drug prescriptions in patients with swallowing difficulties is not optimal, and may even have iatrogenic effects. In this study, 12.7% of the modifications of the drug form could have been harmful. Doctors, pharmacists and nurses need to reevaluate their practices.

Progress and challenges of engineering a biophysical CO2-concentrating mechanism into higher plants.

Growth and productivity in important crop plants is limited by the inefficiencies of the C3 photosynthetic pathway. Introducing CO2-concentrating mechanisms (CCMs) into C3 plants could overcome these limitations and lead to increased yields. Many unicellular microautotrophs, such as cyanobacteria and green algae, possess highly efficient biophysical CCMs that increase CO2 concentrations around the primary carboxylase enzyme, Rubisco, to enhance CO2 assimilation rates. Algal and cyanobacterial CCMs utilize distinct molecular components, but share several functional commonalities. Here we outline the recent progress and current challenges of engineering biophysical CCMs into C3 plants. We review the predicted requirements for a functional biophysical CCM based on current knowledge of cyanobacterial and algal CCMs, the molecular engineering tools and research pipelines required to translate our theoretical knowledge into practice, and the current challenges to achieving these goals.

Role of pulsatile perfusion with tissue plasminogen activator in deceased donor kidneys with extensive glomerular thrombosis.

BACKGROUND: Pulsatile perfusion (PP) improves delayed graft function, whereas tissue plasminogen activator (tPA) lyses thrombi. We studied the role of PP with tPA containing perfusate in deceased donor kidneys (DDK) with 50% thrombosed glomeruli. METHODS: Fourteen DDK with extensive glomerular thrombi on biopsies were preserved using PP with histidine-tryptophan-ketoglutarate solution containing tPA. Wedge biopsies were repeated after PP. RESULTS: Causes of donor death included closed head trauma in 8, anoxia in 2, and stroke in 4. Donors who averaged 33.3 years old displayed a final 24-hour urine volume of 1933 mL, a terminal serum creatinine level of 1.8 mg/dL, a blood urea nitrogen of 20 mg/dL, and a platelet count of 128,000/microL. The initial flow of 47 mL improved to 111 mL/min after 16.17 hours of perfusion. Resistive indices (RI) decreased from 0.69 to 0.26 at 4.2 degrees C. Biopsy specimens after PP showed a reduction in glomerular thrombi from 50% to 23%. Recipients averaged 54.9 years old. Cold ischemia time averaged 35.5 hours. One patient displayed primary allograft nonfunction, 3 required transient dialysis, and 10 showed prompt allograft function. Recipient follow-up averaged 12 months, with serum creatinine levels of 4.3 mg/dL at 1 week, 2.0 mg/dL at 1 month, and 1.6 mg/dL at last follow-up. CONCLUSIONS: Renal allografts with extensive glomerular thrombosis improved their biopsy appearance following PP with tPA. Improvement in PP parameters allowed successful transplantation of such kidneys that otherwise would have been discarded. PP with tPA appears to be beneficial for kidneys with extensive glomerular thrombosis.

Orthotopic kidney retransplantation in simultaneous pancreas kidney transplant patients with renal failure.

Traditionally transplant nephrectomy was required as a separate procedure prior to retransplantation in simultaneous pancreas kidney transplant patients. An alternative approach combining both procedures is described, during which the rejected kidney is removed and replaced orthotopically by the new allograft.

Ipsilateral portal enteric drained pancreas-kidney transplantation: a novel technique.

In conventionally described simultaneous pancreas-kidney transplantation with portoenteric drainage, renal revascularization is derived from the left iliac vessels and pancreatic revascularization, from the right iliac artery. A newer technique utilizing the right iliac artery as a single inflow to both organs is described in six patients herein. The technique is less time-consuming and tedious than the standard method, which involves dissection of both iliac vessels. Advantages include shortened dissection time and preservation of the contralateral side for future use.

The cost of steroid-free protocols in primary cadaver kidney transplantation.

Steroid-free immunosuppression regimens have been enjoying recent success in clinical transplantation. The use of antibodies required for such protocols can be an economic burden. We proposed to study their cost in our center. This retrospective study involved 147 consecutive patients subjected to 4 protocols of immunosuppression. The first received triple therapy. The second group received induction with basiliximab, whereas the third received Basiliximab plus cyclosporine (CSA) plus mycophenolate mofetil (MMF), and the fourth received Thymoglobulin plus CSA plus MMF in conjuction with only 4 days of steroid. Rejection episodes were treated with Solumedrol. Six-month charges were obtained from computerized records of the finance department, the in-house laboratories, and the transplantation service registry. All charges were expressed in 2004 dollars. Statistical analyses were obtained using chi-square, analysis of variance (ANOVA) and Kaplan-Meier tests. The 4 groups were similar with regard to donor and/or recipient gender, race, panel reactive antibodies, cold ischemia, dialysis requirements length of stay and readmission, graft survival, and function. Charges were significantly higher in the last 2 groups as compared with triple therapy.

The "fatty pancreas allograft": anatomopathologic findings and clinical experience.

Thirty-three hematoxylin and eosin-stained sections from 7 turned down pancreata from donors with body mass index (BMI) of 33.3 (range, 28-42) were examined. There was more fat infiltration between the superficial pancreatic lobules, interspersed with superficial islands of pancreatic tissue with its vessels and ductules. Thicker fatty interlobular septa were observed with numerous intralobular fat vacuoles. One organ had widespread autolysis. No microthrombi were noted. One pancreas from a 24-year-old donor with BMI of 30 was transplanted successfully. The "fatty pancreas" can be defatted and used for transplantation.

The impact of introducing laparoscopic donor nephrectomy to an established renal transplant program.

BACKGROUND: Although the advent of hand-assisted laparoscopic donor nephrectomy (HLDN) has had a positive impact on the donor pool, there is still some concern about its safety. The aim of this study was to assess the impact of a change in surgical access to live-donor nephrectomy on donor-related complication rates, the renal function of the donor, and the graft function of the recipient. METHODS: At our hospital, HLDN was introduced in 1998. Thereafter, we compared 49 consecutive donors undergoing open donor nephrectomy (ODN) between 1987 and 2002 with 57 consecutive donors undergoing HLDN between 1998 and 2002. Donor renal and recipient graft functions were assessed by measuring creatinine levels and urine output, with the addition of warm and cold ischemia time and dialysis requirements in the latter group. Data are presented as means (+/-SD) and analyzed with the Student t-test or Fisher's exact test. RESULTS: The ODN and HLDN donors were comparable for age, gender, body mass index, renovascular anatomy, and preoperative creatinine. Estimated blood loss (370 +/- 280 vs 168 +/- 160 ml, p < 0.0001), time to resumption of oral intake (1.7 +/- 0.5 vs 1.3 +/- 0.7 days, p = 0.01), duration of intravenous narcotic requirements (23 +/- 0.7 vs 1.7 +/- 1.0 days, p < 0.0001), and hospital stay (4.2 +/- 1.4 vs 2.9 +/- 1.3 days, p < 0.0001) were significantly decreased after HLDN. There were no significant differences between ODN and HLDN in operating time (204 +/- 46 vs 202 +/- 49 min), donor-related complication rates (12.2% vs 14%), or donor renal and recipient graft functions. CONCLUSION: The introduction of HLDN to an established renal transplant program led to an improved short-term outcome without any increase in donor-related complication rates or delay in recipient graft function.

p53 and Fas ligand are required for psoralen and UVA-induced apoptosis in mouse epidermal cells.

A combination of 8-methoxypsoralen (8-MOP) and ultraviolet-A (UVA) radiation (320-400 nm) (PUVA) is widely used in the treatment of psoriasis and other skin diseases. PUVA is highly effective in eliminating hyperproliferative cells in the epidermis, but its mechanism of action has not been fully elucidated. In this study, we used immortalized JB6 mouse epidermal cells, p53(-/-), and Fas ligand deficient (gld) mice to investigate the molecular mechanism by which PUVA induces cell death. The results indicate that PUVA treatment induces apoptosis in JB6 cells. In addition, PUVA treatment of JB6 cells results in p53 stabilization, phosphorylation, and nuclear localization as well as induction of p21(Waf/Cip1) and caspase-3 activity. In vivo studies reveal that PUVA treatment induces significantly less apoptosis in the epidermis of p53(-/-) mice compared to p53(+/+) mice. Furthermore, FasL-deficient (gld) mice are completely resistant to PUVA-induced apoptosis compared to wild-type mice. These results indicate that PUVA treatment induces apoptosis in mouse epidermal cells in vitro and in vivo and that p53 and Fas/Fas ligand interactions are required for this process, at least in vivo. This implies that similar mechanisms may be involved in the elimination of psoriatic keratinocytes from human skin following PUVA therapy.

Ultraviolet a radiation suppresses an established immune response: implications for sunscreen design.

The ultraviolet radiation present in sunlight is the primary cause of nonmelanoma skin cancer and has been implicated in the development of cutaneous malignant melanoma. In addition, ultraviolet is immune suppressive and the suppression induced by ultraviolet radiation has been identified as a risk factor for skin cancer induction. Ultraviolet also suppresses the immune response to infectious agents. In most experimental models, ultraviolet is applied to immunologically naive animals prior to immunization. Of equal concern, however, is the ability of sunlight to suppress established immune reactions, such as the recall reaction in humans, which protects against microbial infections. Here we demonstrate that solar-simulated ultraviolet radiation, applied after immunization, suppresses immunologic memory and the elicitation of delayed-type hypersensitivity. Further, we found that wavelengths in the ultraviolet A region of the solar spectrum were critical for inducing immune suppression. Ultraviolet A (320-400 nm) radiation was as effective as solar-simulated ultraviolet A + B (290-400 nm) in suppressing the elicitation of an established immune response. Irradiation with ultraviolet AI (340-400 nm) had no effect. Supporting a critical role for ultraviolet A in ultraviolet-induced immune suppression was the observation that applying a sunscreen that contained an ultraviolet B only filter had no protective effect, whereas, a sunscreen containing both ultraviolet A and ultraviolet B filters totally blocked ultraviolet-induced immune suppression. These data suggest that sunlight may depress the protective effect of prior vaccination. In addition, the observation that ultraviolet A is immunosuppressive indicates the need for ultraviolet A protection when designing sun protection strategies.

Postponed aging and desiccation resistance in Drosophila melanogaster.

Studies with the fruit fly, Drosophila melanogaster, have repeatedly shown that selection for postponed reproduction leads to increases in mean life span and increased stress resistance; including increased resistance to desiccation, starvation and ethanol vapors. We show that desiccation resistance declines with age in both short- and long-lived flies suggesting that desiccation resistance may serve as a useful biomarker for aging-related declines in physiological performance. We examined the physical basis of desiccation resistance in five replicate populations selected for postponed reproduction and five replicate control populations. The variables examined were water content, rates of water loss during desiccation, and water content at time of death due to desiccation. In the absence of desiccation stress, both the flies exhibiting postponed senescence and their controls maintained constant water content throughout their lifetimes. In the presence of desiccation stress, the short-lived flies showed significantly higher rates of water loss at all ages than did the long-lived flies. Flies from the two treatments did not differ in water content at death. Our results indicate that water loss rates are the major determinant of desiccation resistance. Water loss rates are under genetic control and covary with age in populations with genetically-determined postponed senescence.