A genetic selection for Mycobacterium smegmatis mutants tolerant to killing by sodium citrate defines a combined role for cation homeostasis and osmotic stress in cell death.

Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine Mycobacterium smegmatis survival in the presence of the chelator sodium citrate. We observed that instead of restricting M. smegmatis growth, concentrated sodium citrate killed M. smegmatis. RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill M. smegmatis under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, mgtE, treZ, and fadD6, we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen M. tuberculosis. Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When Mycobacterium smegmatis is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the mgtE magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis.

Development of chondrocyte-laden alginate hydrogels with modulated microstructure and properties for cartilage regeneration.

Alginate hydrogel is an attractive biomaterial for cell microencapsulation. The microarchitecture of hydrogels can regulate cellular functions. This study aims to investigate the applicability of sodium citrate buffer (SCB) as a culture medium supplement for modulating the microstructure of alginate microbeads to provide a favorable microenvironment for chondrogenic induction. The chondrocyte-laden microbeads, with and without TGF-ß3 incorporation, were produced through an encapsulator. The obtained small-sized microbeads (~300 µm) were exposed to a treatment medium containing SCB, composed of varied concentrations of sodium citrate (1.10-1.57 mM), sodium chloride (3.00-4.29 mM), and ethylenediaminetetraacetic acid (0.60-0.86 mM) to partially degrade their crosslinked structure for 3 days, followed by culture in a normal medium until day 21. Scanning electron microscope micrographs demonstrated a loose hydrogel network with an enhanced pore size in the SCB-treated microbeads. Increasing the concentration of SCB in the treatment medium reduced the calcium content of the microbeads via a Na+ /Ca2+ exchange process and improved the water absorption of the microbeads, resulting in a higher swelling ratio. All the tested SCB concentrations were non-cytotoxic. Increases in aggrecan and type II collagen gene expression and their corresponding extracellular matrix accumulation, glycosaminoglycans, and type II collagen were vividly detected in the TGF-ß3-containing microbeads with increasing SCB concentrations in the treatment medium. Our findings highlighted that the combination of SCB treatment and TGF-ß3 incorporation in the chondrocyte-laden microbeads is a promising strategy for enhancing cartilage regeneration, which may contribute to a versatile application in cell delivery and tissue engineering.

Treatment Effect of Regional Sodium Citrate Anticoagulation in Elderly Patients With High-Risk Bleeding Receiving Continuous Renal Replacement Therapy.

OBJECTIVE: To investigate the safety and efficacy of regional citrate anticoagulation (RCA) on elderly patients at high risk of bleeding after continuous renal replacement therapy (CRRT). METHODS: A total of 31 patients at high risk of bleeding who received CRRT in the intensive care unit were collected. The patients were divided into RCA group (n = 17) and no anticoagulation group (NA, n = 14) according to whether RCA was used or not. The levels of creatinine (Cr), blood urea nitrogen (BUN), prothrombin time (PT), activated partial thromboplastin time (APTT), total calcium (tCa), ionized calcium ion (iCa2+), sodium ion (Na+), bicarbonate ion (HCO3-), tCa/iCa2+ ratio, and pH were observed after treatment. The filter use time, number of filters used, filter obstruction events, clinical outcomes, and safety evaluation indexes were compared post-treatment. RESULTS: After treatment, serum Cr and BUN levels, APTT and PT levels in the RCA group were significantly lower than the NA group. The tCa, iCa2+, HCO3-, tCa/iCa2+, and pH were within the normal range after RCA treatment while Na+ levels saw a significant increase. In the RCA group, the filter using time was significantly longer, with significantly reduced numbers of filter use within 72 h and filter disorder events. Additionally, patients in the RCA group showed significant recovery of renal function and a significant reduction in bleeding events and in-hospital mortality. CONCLUSION: RCA treatment significantly improves clinical outcome of patients at high risk of bleeding after CRRT, safely and effectively prolongs the filter life and avoids coagulation incidences.

Depuration of cadmium from Chlamys farreri by ZnSO4, EDTA-Na2 and sodium citrate in short time.

In view of high content of cadmium (Cd) in Chlamys farreri, a commercial edible shellfish species, depurating Cd from Chlamys farreri is an important topic nowadays, especially in short time. Therefore, three kinds of additives were introduced into seawater respectively, i.e. ZnSO4, EDTA-Na2, sodium citrate, to depurate Cd from Chlamys farreri. The alteration of Cd content in separate organs was investigated under several treatments with high depuration efficiency. The results showed that Cd was depurated exceeding 20% within 12 h by the combination of 0.15 g/L sodium citrate, 0.28 g/L ZnSO4, and 0.42 g/L EDTA-Na2. No obvious increase of Cd was observed in the adductor muscles, while Cd decreased in the other part, so the reduction of Cd in the whole organism of Chlamys farreri may occur. Cd reduction was found in the following organs: the digestive gland, kidney, gill, and mantle. Furthermore, Cd migration to gonad from other tissues was noticed.

Ectoine production with indigenous Marinococcus sp. MAR2 isolated from the marine environment.

Ectoine has fostered the development of products for skin care and cosmetics. In this study, we employed the marine bacterial strain Marinococcus sp. MAR2 to increase ectoine production by optimizing medium constituents using Response Surface Methodology (RSM) and a fed-batch strategy. The results from the steepest ascent and central composite design indicated that 54 g/L of yeast extract, 14.0 g/L of ammonium acetate, 74.4 g/L of sodium glutamate, and 6.2 g/L of sodium citrate constituted the optimal medium with maximum ectoine production (3.5 g/L). In addition, we performed fed-batch culture in the bioreactor, combining pH and dissolved oxygen to produce ectoine by Marinococcus sp. MAR2. The ectoine production, content, and productivity of 5.6 g/L, 10%, and 3.9 g/L/day were further reached by a fed-batch culture. Thus, the ectoine production by Marinococcus sp. MAR2 using RSM and fed-batch strategy shows its potential for industrial production.

Structural and binding studies of phosphopantetheine adenylyl transferase from Acinetobacter baumannii.

Phosphopantetheine adenylyltransferase (PPAT, EC. 2.7.7.3) catalyzes an essential step in the reaction that transfers an adenylyl group from adenosine tri phosphate (ATP) to 4'-phosphopantetheine (pPant) yielding 3'- dephospho-coenzyme A (dPCoA) and pyrophosphate (PP) in the coenzyme A (CoA) biosynthesis pathway. The enzyme PPAT from Acinetobacter baumannii (AbPPAT) was cloned, expressed and purified. The binding studies of AbPPAT were carried out with two compounds, tri­sodium citrate (TSC) and l-ascorbic acid (LAA, vitamin-C) using fluorescence spectroscopic (FS) and surface Plasmon resonance (SPR) methods. Both methods provided similar values of dissociation constants for TSC and LAA which were of the order of 10-8 M and 10-5 M respectively. The computer aided docking studies indicated fewer interactions of LAA with AbPPAT as compared to those of TSC. The freshly purified samples of AbPPAT were crystallized. The crystals of AbPPAT were soaked in the solutions containing TSC and LAA. However, the crystals of the complex of AbPPAT with LAA did not diffract well and hence the structure of the complex of AbPPAT with LAA could not be determined. On the other hand, the crystals of the complex of AbPPAT with TSC diffracted well and the structure was determined at 1.76 Šresolution. It showed that TSC bound to AbPPAT at the ATP binding site and formed several intermolecular contacts including 12 hydrogen bonds. The results of binding studies for both TSC and LAA and the structure of the complex of AbPPAT with TSC clearly indicated a potential role of TSC and LAA as antibacterial agents.