High Osmolarity Research Articles

Dependence of diffusion in Escherichia coli cytoplasm on protein size, environmental conditions, and cell growth.

Inside prokaryotic cells, passive translational diffusion typically limits the rates with which cytoplasmic proteins can reach their locations. Diffusion is thus fundamental to most cellular processes, but the understanding of protein mobility in the highly crowded and non-homogeneous environment of a bacterial cell is still limited. Here, we investigated the mobility of a large set of proteins in the cytoplasm of Escherichia coli, by employing fluorescence correlation spectroscopy (FCS) combined with simulations and theoretical modeling. We conclude that cytoplasmic protein mobility could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. We observed similar size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli. For the faster-diffusing proteins, this size dependence is well consistent with the Stokes-Einstein relation once taking into account the specific dumbbell shape of protein fusions. Pronounced subdiffusion and hindered mobility are only observed for proteins with extensive interactions within the cytoplasm. Finally, while protein diffusion becomes markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity, all of these perturbations affect proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity.

Strain-dependent differences in coordination of yeast signalling networks.

The yeast mitogen-activated protein kinase pathways serve as a model system for understanding how network interactions affect the way in which cells coordinate the response to multiple signals. We have quantitatively compared two yeast strain backgrounds YPH499 and ∑1278b (both of which have previously been used to study these pathways) and found several important differences in how they coordinate the interaction between the high osmolarity glycerol (HOG) and mating pathways. In the ∑1278b background, in response to simultaneous stimulus, mating pathway activation is dampened and delayed in a dose-dependent manner. In the YPH499 background, only dampening is dose-dependent. Furthermore, leakage from the HOG pathway into the mating pathway (crosstalk) occurs during osmostress alone in the ∑1278b background only. The mitogen-activated protein kinase Hog1p suppresses crosstalk late in an induction time course in both strains but does not affect the early crosstalk seen in the ∑1278b background. Finally, the kinase Rck2p plays a greater role suppressing late crosstalk in the ∑1278b background than in the YPH499 background. Our results demonstrate that comparisons between laboratory yeast strains provide an important resource for understanding how signalling network interactions are tuned by genetic variation without significant alteration to network structure.

Genome Analysis of Halomonas elongata Strain 153B and Insights Into Polyhydroxyalkanoate Synthesis and Adaptive Mechanisms to High Saline Environments.

Species of the Halomonas genus are gram-negative, aerobic, moderately halophilic bacteria that synthesize polyhydroxyalkanoates (PHAs) and other high-value products that have a wide range of potential uses in the food, feed, cosmetics, pharmaceutical, and chemical sectors. Genome sequencing studies allow for the description and comparison of genetic traits with other strains and species, allowing for the exploration of the organism's potential, necessary to further biotechnology applications. Here, the genome of Halomonas elongata strain 153B was sequenced, its features compared to 5 other strains and 7 species, and a description of features for adaptations to hypersaline environments and bioproducts synthesis was done. Whole-genome analysis showed H. elongata 153B has more similar features to the reference strain H. elongata DSM 2581 compared to 4 other reported strains. Comparative genomics showed 2064 core genomic clusters between the strains and 666 singletons for strain 153B. Several genes in transport and signaling, osmoregulation, and oxidative stress that have roles in adaptation to environments with high osmolarity were also revealed. These appear to form an intricate network of overlapping systems carefully coordinated to bring about adaptation. H. elongata 153B genes for the synthesis of PHAs, ectoine, vitamins, and the degradation of drugs and aromatic compounds were described. The results will aid in the study of halophile physiology, provide a mine for valuable enzymes, and help speed up research for other biotechnology applications.

A Stress Hub in Scedosporium apiospermum: The High Osmolarity Glycerol (HOG) Pathway

Scedosporium species are opportunistic filamentous fungi found in human-impacted areas. Clinically relevant species, such as S. apiospermum, rank as the second most frequent colonizers of the airways of patients with cystic fibrosis (CF), which are characterized by persistent oxidative stress. This raises the question of how Scedosporium species abate conditions imposed in hostile environments. Since the High Osmolarity Glycerol (HOG) pathway plays a central role in fungal adaptation to stress, we aimed to pheno-profile the involvement of the pathway in response to stress in S. apiospermum using Western blot. We show for the first time that a wide range of stress distinctively activates the HOG pathway in S. apiospermum, including oxidants (H2O2, menadione, cumene hydroperoxide, diamide, paraquat, and honokiol), osmotic agents (sorbitol and KCl), cell-wall stress agents (caffeine, calcofluor white, and Congo Red), heavy metals (cadmium and arsenite), fungicides (fludioxonil and iprodione), antifungals (voriconazole and amphotericin B), and acid stress (pH 4). We suggest that the function of the HOG pathway as a general stress regulator is also conserved in S. apiospermum.

Safety of peripherally administered 3% hypertonic saline

BackgroundAdministration of 3% sodium chloride through a peripheral venous catheter is associated with risk of infusion-related adverse events (IRAE) due to its high osmolarity. Given this concern and the paucity of data regarding these events, many hospitals have policies that require central line administration of 3% sodium chloride. ObjectiveThe objective of this analysis was to evaluate the incidence of IRAE associated with peripheral administration of 3% sodium chloride. MethodsThis analysis included patients who received 3% sodium chloride via a peripheral venous catheter between May 2017 and August 2019. The major endpoint of this analysis was the overall incidence of IRAE, defined as the documentation of infiltration or phlebitis. A multivariable logistic regression was performed to identify potential risk factors (e.g., age, infusion rate, infusion duration, peripheral venous catheter location, and needle gauge) for development of IRAE. ResultsA total of 706 administrations in 422 patients were included. Seventy-four (10.5%) administrations were associated with a documented event. Based on the Infusion Nurses grading scale for infiltration or phlebitis, 48% of the events in this analysis were grade 1 in severity. Duration of infusion of 3% sodium chloride was found to be associated with an increased odds of an IRAE (OR per 1 h 1.02, 95% CI 1.01–1.02) in the multivariable analysis. Age, infusion rate, peripheral venous catheter location, and needle gauge were not independently associated with an increased risk of an IRAE. ConclusionThese data suggest that IRAE occurred more frequently when 3% sodium chloride was administered over a longer duration and the majority of events were mild with no permanent tissue injury. It may be reasonable to consider peripheral administration of 3% sodium chloride in the acute care setting for a short duration, although additional studies are needed to continue to evaluate its safety.

PSUN242 Severe Hypernatremia in a patient with combined Hyperosmolar Hyperglycemic State and Diabetic Ketoacidosis

Abstract Introduction Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS) are the complications of diabetes with highest morbidity. In both, there are fluid shifts and osmotic diuresis with electrolyte disturbances that result in hyponatremia. With high glucose levels, sodium become falsely decreased due to dilutional effects of water shifting from intracellular to extracellular space. Hypernatremia is an uncommon electrolyte abnormality in this setting, with unclear exact etiology. We present a 35-year-old gentleman who was found to have severe hypernatremia in the setting of combined DKA and HHS. Clinical Case A 35-year-old male with class III obesity presented to the emergency department with altered sensorium and lethargy. Patient was having nausea and vomiting, along with polyuria, polydipsia and nocturia, prior to worsening encephalopathy. Physical exam showed rapid shallow breathing and lethargy. No focal neurologic deficits noted.Initial labs revealed severe hyperglycemia and hypernatremia with sodium at 164 mmol/L, severe metabolic acidosis, and acute kidney injury. Hemoglobin A1C was 9.8%. Ketonuria and ketonemia were present. Given the high osmolality, he was diagnosed with DKA and HHS.Free water deficit was 15.4 L. Insulin drip was started, along with 5 liters of intravenous Lactated Ringer's boluses. As blood glucose improved, patient was placed on dextrose-5% water infusion at 200 mL/hour.Encephalopathy improved approximately 48 hours after admission. His glucose stabilized, renal function improved, acidosis resolved, and sodium returned to normal. Sodium was corrected at a rate of 8 mmol/L daily. He was transitioned to general wards and started on an oral carbohydrate-restricted diet and subcutaneous insulin regimen containing long- and short-acting insulin analogs. Conclusion During severe hyperglycemia, glucose increases extracellular osmolality, creating a gradient for water to move from intracellular to extracellular compartments, leading to hyponatremia. As the renal threshold is reached, glucosuria ensues, causing osmotic diuresis and water loss. If this process continues, excess water loss can cause dehydration, further increase serum osmolality, and lead to hypernatremia.A previous study evaluated patients who present with hypernatremia during DKA. It was found that patients with new-onset diabetes are more likely to present with hypernatremia (1).Several etiologies contribute to altered sensorium in such patients, including cerebral hypoperfusion in the setting of dehydration and excess water loss, acidosis, and direct effect of ketone bodies on brain cells (2). As electrolyte abnormalities are corrected with adequate fluid selection, patients return to baseline mentation. It is best to correct hypernatremia at a rate of 6-8 mmol/L daily to prevent the complications of rapid correction. References KW Monroe, WD King, MH Nichols, JA Atchinson. Hypernatremia in diabetic ketoacidosis. Practical Diabetes Int 1997;14(6): 159-61. Elias Estifan, Sushant M. Nanavati, Vinod Kumar, et al. Salty diabetes: a case series of hypernatremia presenting with diabetic ketoacidosis. AME Case Rep 2019;3: 27. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

RF25 | PMON113 Subarachnoid Hemorrhage Complicated by Nephrogenic and Central Diabetes Insipidus

Abstract Introduction Diabetes insipidus (DI) is a rare complication of subarachnoid hemorrhage (SAH) and associated with worse prognosis and increased mortality. We present a case of combined nephrogenic and central DI together with osmotic diuresis as a result of SAH and discuss the pathophysiology. Case A 63-year-old female without significant past medical history was found unresponsive. CT head followed by angiography confirmed extensive SAH arising from a saccular anterior communicating artery (Acoma) aneurysm. She underwent emergent coil embolization and right frontal external ventriculostomy drain placement. Postoperatively, she developed polyuria (300-400 mL/hour) and hypernatremia (156 mEq/L) concerning for the development of DI. Serum osmolality was elevated at 316 mOsm/kg, urine osmolality inappropriately low relative to serum osmolality at 289 mOsm/kg but also unexpectedly high for complete DI, serum potassium 2.9 mEq/L and urine potassium elevated urine at 9 mEq/L. Serum copeptin was 6 pmol/L and inappropriately low for nephrogenic DI, suggesting a mixed central and peripheral process as well as contribution from osmotic diuresis. Intravenous (IV) desmopressin (DDAVP) only slightly reduced the polyuria. Management was complicated by adypsia and persistent hypokalemia requiring ongoing replacement. IV fluid, which had initially consisted of high-osmolality Plasma-Lyte (294 mOsm/L), matched to urine output, was switched to D5W, leading to a significant decrease in polyuria. On post-operative day 17, DDAVP was discontinued with normalization of sodium (137 mEq/L), potassium (3.8 mEq/L) and urine output. Three months post-operatively, she was asymptomatic with intact pituitary function and no hypokalemia. Discussion Hypokalemia in patients with SAH is thought due to acute intracellular shift of potassium caused by excessive β2-adrenergic activity generated endogenously by the sympathetic nervous system. In our patient, there was also likely contribution from distal tubular delivery of sodium from osmotic diuresis, leading to increased urinary potassium losses and hypokalemia-induced partial nephrogenic DI which was further complicated by partial central DI as suggested by the inappropriately low copeptin level for nephrogenic DI in face of a high serum osmolality and inappropriately low urine osmolality. As the vascular supply to anterior hypothalamus is derived from the Acoma, rupture may compromise perfusion affecting neuronal populations in the lamina terminalis that regulate thirst and AVP secretion in response to hyperosmolarity. Early detection of DI is critical because hypovolemia can predispose to cerebral vasospasm and worsen outcomes. This case also depicts the importance of careful consideration of sources of osmolar loads such as the composition of administered IV fluids, and aggressive potassium replacement to maintain DDAVP sensitivity. Conclusion DI should be considered in polyuria syndrome with hypernatremia after a neurosurgical event. Nephrogenic and central DI and adypsia can manifest following SAH, with sources of iatrogenic osmolar load further complicating the clinical picture. Early recognition and treatment may reduce morbidity and mortality. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:12 p.m. - 1:17 p.m.

PMON318 Nephrogenic syndrome of inappropriate antidiuresis in a 19 months old child with gross developmental delay

Abstract Case report 19 months old African American boy with nonsignificant past medical history presented with inability to walk, hypotonia chewing difficulties and speech delay. Initial laboratory evaluations demonstrated serum hyponatremia of 120-129 mEq/dl, low serum osmolality 261 mosm/kg (normal serum osmolality 275-295 mosm/kg) with inappropriately high urine osmolality 445 mosm/kg) (normal urine osmolality 300-1000 mosm/kg). Blood work for thyroid, adrenal, and pituitary function was unremarkable. Vasopressin level of 2.4 pg/ml (normal 0.0 - 4.7 pg/mL) did not support the diagnosis of Syndrome of Inappropriate Antidiuretic Hormone Secretion (SIADH). Fludrocortisone challenge test ruled out aldosterone deficiency with insufficient decrease in urine sodium and no response in serum sodium. Renal ultrasound and brain MRI were normal. Genetic testing revealed AVPR2 X-linked mutation c.409C>T p.R137C, consistent with nephrogenic syndrome of inappropriate antidiuresis (NSIAD). The patient was discharged on sodium chloride tablets 2g every 8 hours and remained stable, asymptomatic. One month after discharge patient showed significant neurological improvement. Discussion Sodium is known to play a significant role in growth and maturation of the whole body, especially of the nervous system. Moderate (120-129 mEq/L) to severe (<120 mEq/L) hyponatremia can present with nausea, malaise, headache, lethargy, motor and speech delay, seizures and even death due to brain herniation. Normal fluid and electrolyte balance is achieved by the thirst mechanism and free water excretion in kidneys. This is mediated by Vasopressin, which acts through V2R receptor in distal renal collecting duct. Syndrome of inappropriate antidiuretic hormone secretion (SIADH) is the most common cause of hyponatremia. Reset osmostat syndrome with a congenitally low serum osmolality threshold is one of the rare disorders on the differential. Hereditary nephrogenic syndrome of inappropriate antidiuresis is very rare mutation in vasopressin receptor, which clinically resembles SIADH except for normal/low vasopressin levels in set of hyponatremia. It causes gain of function and inappropriately high water retention. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

PSUN235 Dopamine-induced polyuria

Abstract Background Dopamine acts on dopamine-1 receptors in the renal vasculature at a low dose, resulting in natriuresis and diuresis. Polyuria is a rare condition found in patients who receive dopamine infusion. We report a young female hospitalized for ovarian torsion who underwent several surgical procedures had been infected with gram-negative bacteremia. She received a dopamine infusion for hypotension with relative bradycardia. Subsequently, she developed polyuria. Case presentation A 25-year-old female was referred to UMC hospital for acute diverticulitis with micro-perforation from the sigmoid colon. She presented to the outside hospital with abdominal pain, fever, nausea, and vomiting for four days. On arrival, she had a fever of 101.9F. Initial laboratory showed leukocytosis and lactic acidosis. CT abdomen at UMC revealed 16×11cm mass in the left lower quadrant, potentially originating from the left ovary. The patient was treated with IV Zosyn for acute diverticulitis. On day 5 of admission patient remained to have abdominal pain, fever, and worsening lactic acidosis. She was taken to OR for exploratory laparotomy with dissection of left salpingo-oophorectomy for torsion of adnexal mass resection. On day 91 of admission, the patient developed low-grade fever (100.7F) and hypotension (BP 63/30) with the MAP of 40. IV NS 1L was administered. CT abdomen revealed no intra-abdominal or pelvic abscess or fluid collection. For possible septic shock, the patient was started on empirical IV antibiotics. Despite IV fluid resuscitation, blood pressure remained low, vasopressin and norepinephrine were initiated. Day 92, she developed new-onset bradycardia, EKG showed third-degree AV block. IV dopamine infusion was started at the rate of 5mcg/kg/min. During dopamine infusion, the patient voided between 400-600mL/h. Over the 12-h period, the urine output was about 5L. Endocrinology was consulted for polyuria. Laboratory studies showed urine osmolality 384mOsm/kg, urine electrolytes were UNa 130mmol/L, UK 20.1mmol/L, UCl 115mmol/L, BUN/Cr 28/1.0. Approaching polyuria, the diuresis was associated with high urine osmolality and sodium, which exhibited characteristic features of solute diuresis with natriuresis. The diuretic phase of acute renal injury could not be implicated in this patient since she has stable renal function without azotemia. There was no evidence of urinary tract obstruction to suggest post obstruction diuresis. Central or peripheral diabetes insipidus was ruled out by the lack of characteristic water diuresis. It was suspected that the diuresis was related to the dopamine infusion. Thus, dopamine infusion was discontinued. The urine output dropped within a few hours. The patient's urine output stabilized to 0.8-1.0ml/min. The striking drop in urine output after discontinuity of dopamine is consistent with the role of dopamine in this patient's diuresis. Conclusion This is a rare case report of dopamine-induced polyuria in septic shock patient. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

ODP332 Maternal Central Diabetes Insipidus Immediately after Delivery, Owing to Vasospasm of the Internal Carotid Artery

Abstract Background Sheehan's syndrome is a cause of hypopituitarism during the postpartum period, but it rarely manifests immediately after birth. Clinical Case We report the case of a 30-year-old woman who delivered her second child, lost 1,050 mL of blood in the process, but did not receive a blood transfusion. A few hours after delivery, the patient complained of headache, nausea, and thirst, and her urine production increased to 9 L/day. No menstrual irregularities or abnormalities during pregnancy or childbirth had been reported. None of hypotension, external eye muscle paralysis, or visual impairment were identified on physical examination. The laboratory findings were low urine osmolality and high serum osmolality and sodium concentration. The concentrations of thyroid hormones, growth hormone (GH), and anti-diuretic hormone (ADH) were found to be low. The IgG4 concentration was not high (4.7 mg/dL; normal <105 mg/dL) and the patient was negative for anti rabphilin-3A antibody. Pituitary magnetic resonance imaging (MRI) on the day of admission showed enlargement of the pituitary gland and stalk, and magnetic resonance angiography showed stenosis of the left internal carotid artery. The patient showed a poor response to GH-releasing peptide 2 and thyroid releasing hormone stimulation. Hypertonic saline administration did not cause an increase in ADH concentration. We diagnosed adult growth hormone deficiency (aGHD), central hypothyroidism, and central diabetes insipidus (DI). The enlargement of the pituitary gland was slightly less marked and the stenosis of the left internal carotid artery improved 7 days after admission. After 6 months, the pituitary gland was almost normal in size, although the aGHD, central hypothyroidism, and central DI remained. Sheehan's syndrome or lymphocytic hypophysitis could have been responsible for the postpartum hypopituitarism. This case was not typical of lymphocytic hypophysitis with respect to the hormone deficiency pattern, the MRI findings, and the lack of anti rabphilin-3A antibody. Sheehan's syndrome is caused by ischemic necrosis of the anterior pituitary gland after severe postpartum hemorrhage. The prevalence of central DI is estimated to be 2%–3%. Typically, pituitary dysfunction progresses slowly and develops several years after childbirth. In contrast, in the present patient, stenosis of the left internal carotid artery developed and improved during short periods of time. The vasospasm of the internal carotid artery caused reductions in flow through the superior and inferior hypophysial arteries, which resulted in ischemia and necrosis of the pituitary gland, similar to Sheehan's syndrome. Clinical Lesson: Although rare, maternal central DI can be caused by ischemia and vasospasm immediately after the delivery of a child. The possibility of a pituitary stroke in the presence of neoplastic disease should not be ruled out, and imaging and pituitary gland function should be followed over the long term. Presentation: No date and time listed

Bioactivity profiling of the extremolyte ectoine as a promising protectant and its heterologous production.

Ectoine is a compatible solutes that is diffusely dispersed in bacteria and archaea. It plays a significant role as protectant against various external pressures, such as high temperature, high osmolarity, dryness and radiation, in cells. Ectoine can be utilized in cosmetics due to its properties of moisturizing and antiultraviolet. It can also be used in the pharmaceutical industry for treating various diseases. Therefore, strong protection of ectoine creates a high commercial value. Its current market value is approximately US$1000kg-1. However, traditional ectoine production in high-salinity media causes high costs of equipment loss and wastewater treatment. There is a growing attention to reduce the salinity of the fermentation broth without sacrificing the production of ectoine. Thus, heterologous production of ectoine in nonhalophilic microorganisms may represent the new generation of the industrial production of ectoine. In this review, we summarized and discussed the biological activities of ectoine on cell and human health protection and its heterologous production.

Outer membrane-anchoring enables LpoB to regulate peptidoglycan synthesis rate

Peptidoglycan (PG) is an essential component of the cell envelope in most bacteria, responsible for maintaining the shape of the cell and protecting the cell from environmental stresses. The growth of the PG layer during cell elongation and division is facilitated by the coordinated activities of PG synthases and hydrolases. PG synthases are regulated from inside the cell by components of the elongasome and divisome complexes driven by the cytoskeletal proteins MreB and FtsZ. In Escherichia coli the PG synthases PBP1A and PBP1B require the activation by outer membrane (OM)-anchored lipoproteins LpoA and LpoB, respectively. These have an elongated structure and are capable to span the periplasm to reach their cognate, cytoplasmic membrane (CM)-anchored PG synthase through the PG layer. Presumably, the Lpo proteins activate the PBPs at sites where the PG mesh is stretched or defective, resulting in coupling of PG synthase activation with cell growth or PG repair. Here we investigated the importance of OM-anchoring on the function of Lpo proteins in regulating PG synthesis in response to environmental stresses. We investigated the effects of an artificially CM-tethered LpoB on cell morphology and PG synthesis. Our results indicate that mis-localization of LpoB affects the growth and morphology of cells in high osmolarity growth medium, and PG synthesis rate upon an osmotic upshift.

Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station.

Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS without directly contacting the embryos. First, we developed several new devices using a hollow fiber tube that allows thawing embryo without practice and observations of embryonic development. The recovery rate of embryos was over 90%, and its developmental rate to the blastocyst were over 80%. However, the general vitrification method requires liquid nitrogen, which is not available on the ISS. Therefore, we developed another new device, Embryo Thawing and Culturing unit (ETC) employing a high osmolarity vitrification method, which preserves frozen embryos at -80°C for several months. Embryos flushed out of the ETC during thawing and washing were protected using a mesh sheet. Although the recovery rate of embryos after thawing were not high (24%-78%) and embryonic development in ETC could not be observed, thawed embryos formed blastocysts after 4 days of culture (29%-100%) without direct contact. Thus, this ETC could be used for untrained astronauts to thaw and culture frozen embryos on the ISS. In addition, this ETC will be an important advance in fields such as clinical infertility and animal biotechnology when recovery rate of embryos were improved nearly 100%.

The very long-chain fatty acid elongase FgElo2 governs tebuconazole sensitivity and virulence in Fusarium graminearum.

Very long-chain fatty acids (VLCFAs), the precursors for the synthesis of sphingolipids (SLs), play pivotal roles in the development and stress response in eukaryotes. In Saccharomyces cerevisiae, VLCFAs are synthesized in the endoplasmic reticulum (ER) through a four-step elongation cycle. However, the functions of VLCFA elongases in phytopathogenic fungi remain largely unexplored. Here, we identified a single copy of the VLCFA elongase gene FgELO2 in Fusarium graminearum that causes Fusarium head blight worldwide. FgElo2 localized to ER membrane, and deletion mutant of FgELO2 exhibited serious defects in vegetative growth and conidiation. Importantly, ΔFgElo2 led to ergosterol content reduction and disrupted the ER-localization of 14-α-demethylase FgCyp51s, indicating that the scarce of SLs reduced ergosterol, which ultimately elevated the sensitivity of ΔFgElo2 to tebuconazole. Fluorescent microscopic examination suggested that FgElo2 was degraded upon cell membrane stress. ΔFgElo2 showed decreased phosphorylation of high osmolarity glycerol (HOG) pathway and subsequently exhibited remarkable sensitivity to osmotic stress. In addition, fungal virulence was dramatically reduced in ΔFgElo2 via inhibiting deoxynivalenol production and formation of infection structures. Together, this study demonstrates that the VLCFA elongase FgElo2 modulates fungal development, tebuconazole sensitivity, stress responses and virulence, which may advance our understanding of pathogen-host interactions mediated by VLCFAs.

OmpR and Prc contribute to switch the Salmonella morphogenetic program in response to phagosome cues.

Salmonella enterica serovar Typhimurium infects eukaryotic cells residing within membrane-bound phagosomes. In this compartment, the pathogen replaces the morphogenetic penicillin-binding proteins 2 and 3 (PBP2/PBP3) with PBP2SAL /PBP3SAL , two proteins absent in Escherichia coli. The basis for this switch is unknown. Here, we show that PBP3 protein levels drop drastically when S. Typhimurium senses acidity, high osmolarity and nutrient scarcity, cues that activate virulence functions required for intra-phagosomal survival and proliferation. The protease Prc and the transcriptional regulator OmpR contribute to lower PBP3 levels whereas OmpR stimulates PBP2SAL /PBP3SAL production. Surprisingly, despite being essential for division in E. coli, PBP3 levels also drop in non-pathogenic and pathogenic E. coli exposed to phagosome cues. Such exposure alters E. coli morphology resulting in very long bent and twisted filaments indicative of failure in the cell division and elongation machineries. None of these aberrant shapes are detected in S. Typhimurium. Expression of PBP3SAL restores cell division in E. coli exposed to phagosome cues although the cells retain elongation defects in the longitudinal axis. By switching the morphogenetic program, OmpR and Prc allow S. Typhimurium to properly divide and elongate inside acidic phagosomes maintaining its cellular dimensions and the rod shape.

A test for plasticity in sperm motility activation in response to osmotic environment in an anuran amphibian.

Evolutionary theory predicts that selection will favor phenotypic plasticity in sperm traits that maximize fertilization success in dynamic fertilization environments. In species with external fertilization, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but evidence for osmotic‐induced sperm plasticity is limited to euryhaline fish and marine invertebrates. Whether this capacity extends to freshwater taxa remains unknown. Here, we provide the first test for plasticity in sperm‐motility activation in response to osmotic environment in an anuran amphibian. Male common eastern froglets (Crinia signifera) were acclimated to either low (0 mOsmol kg−1) or high (50 mOsmol kg−1) environmental osmolality, and using a split‐sample experimental design, sperm were activated across a range of osmolality treatments (0, 25, 50, 75, 100, and 200 ± 2 mOsmol kg−1). Unexpectedly, there was no detectable shift in the optimal osmolality for sperm‐motility activation after approximately 13 weeks of acclimation (a period reflecting the duration of the winter breeding season). However, in both the low and high acclimation treatments, the optimal osmolality for sperm‐motility activation mirrored the osmolality at the natural breeding site, indicating a phenotypic match to the local environment. Previously it has been shown that C. signifera display among‐population covariation between environmental osmolality and sperm performance. Coupled with this finding, the results of the present study suggest that inter‐population differences reflect genetic divergence and local adaptation. We discuss the need for experimental tests of osmotic‐induced sperm plasticity in more freshwater taxa to better understand the environmental and evolutionary contexts favoring adaptive plasticity in sperm‐motility activation.

Guilty by association: Importers, exporters and MscS-type mechanosensitive channels encoded in biosynthetic gene clusters for the stress-protectant ectoine.

Ectoine and its derivative hydroxyectoine are widely synthesized or imported by bacteria to fend off the detrimental effects of high osmolarity on cellular hydration and growth. Genes that are connected to a particular physiological process are often found in the same genomic context. We exploited this feature in a comprehensive bioinformatical analysis of 1103 ectoine biosynthetic gene clusters from Bacteria and Archaea through which we identified 415 ect operons that colocalize with genes encoding potential osmolyte transporters. These belong to various importer families. Focusing on the complex ect gene clusters of the alpha-proteobacteria Hyphomonas neptunium and Novoshingobium sp. LH128, we analysed several transporters with respect to their substrate specificities through physiological, molecular and modelling approaches. Accordingly, we identified an MFS-type uptake system specific for ectoines (EctU) and a novel SSS-type ectoine/hydroxyectoine importer (EctI) with a broader substrate profile for osmostress protectants. Furthermore, some ect gene clusters encode a MscS/YbdG-type mechanosensitive channel protein, whose functionality was assessed through down-shock assays. Moreover, our analysis identified the gene for the first putative ectoine/hydroxyectoine-specific efflux system (EctX), a member of the MFS superfamily. Our findings make substantial contributions to the understanding of the ecophysiology of ectoines, key players in microbial osmostress adjustment systems.

Safety and Tolerability of 23.4% Hypertonic Saline Administered Over 2 to 5 Minutes for the Treatment of Cerebral Herniation and Intracranial Pressure Elevation.

Hyperosmolar therapy is the cornerstone of medical management of sustained elevated intracranial pressure from cerebral edema. Acute intracranial hypertension and herniation is a medical emergency that requires rapid treatment and stabilization to prevent secondary brain injury or death. Intravenous hypertonic sodium chloride (NaCl) 23.4% is an effective treatment modality commonly used in this setting. Because of its high osmolarity, use has historically been limited primarily to central venous line administration as an intermittent infusion due to concerns about thrombophlebitis, injection site pain, and tissue necrosis or injury with extravasation. The objective of this analysis was to prospectively evaluate the safety of administration of 23.4% NaCl as a rapid intravenous push over 2-5min. A prospective analysis of patients admitted between April 2021 and December 2021 who received 23.4% NaCl intravenous push over 2-5min in a central or peripheral line was performed. Safety end points included incidence of new onset hypotension [defined as systolic blood pressure (SBP) < 90mm Hg or SBP decrease of at least 20mm Hg], bradycardia (defined as heart rate < 50 beats per minute), and infusion site reactions documented within 1h of administration. For secondary safety outcomes, highest and lowest SBP and lowest heart rates documented within 1h before 23.4% NaCl administration were compared with values collected within 1h post administration and evaluated by mixed-design analysis of variance test with adjustment for peripheral versus central line administration. We identified 32 patients who received 79 administrations of 23.4% NaCl through a central line or peripheral line during the study period. An SBP decrease of at least 20mm Hg was observed in 13% of patients, an SBP < 90mm Hg occurred in 16% of patients, and bradycardia occurred in 3% of patients who received 23.4% NaCl. Injection site pain was reported by one patient without documented thrombophlebitis, cellulitis, or tissue damage. Pain was not reported during two subsequent administrations in the same patient. There was no documented occurrence of soft tissue injury or necrosis in any patient. Compared with baseline vital signs before 23.4% NaCl administration, no difference in vital signs post administration was observed. Central and peripheral administration of 23.4% NaCl over 2-5min was well tolerated, and incidence of hypotension, bradycardia, or infusion site-related adverse events was rare.

Effect of Metschnikowia pulcherrima on Saccharomyces cerevisiae PDH By-Pass in MixedFermentation with Varied Sugar Concentrations of Synthetic Grape Juice and Inoculation Ratios

The effects of Metschnikowia pulcherrima and high glucose osmolality on S. cerevisiae pyruvate dehydrogenase pathway (PDH) by-pass were examined by varying the starting sugar concentration of synthetic grape juice and the inoculation ratio of S. cerevisiae to M. pulcherrima. The findings revealed that M. pulcherrima and osmolarity impacted S. cerevisiae’s PDH by-pass. The inoculation concentration of M. pulcherrima significantly affected pyruvate decarboxylase (PDC) activity and acs2 expression when the initial sugar concentration was 200 g L−1 and 290 g L−1. The osmolarity caused by the initial sugar (380 g L−1) significantly influenced the enzymatic activity of S. cerevisiae, which decreased PDC and acetaldehyde dehydrogenase (ALD) activities while increasing Acetyl-CoA synthetase (ACS) activity. The reduction in acetic acid in the wine was caused by M. pulcherrima altering the initial sugar concentration faced by S. cerevisiae, which in turn affected enzymatic activity. The alteration of enzyme activity and accumulation of primary metabolites revealed why mixed fermentation could reduce the acetic acid content in wine by altering the enzymatic activity and affecting the expression of several key genes. The M. pulcherrima inoculation levels had no significant effect on the acetic acid and glycerol concentration in the same fermentation medium.

Melatonin loaded hybrid nanomedicine: DoE approach, optimization and in vitro study on diabetic retinopathy model

Melatonin (MEL) is a pleiotropic neurohormone of increasing interest as a neuroprotective agent in ocular diseases. Improving the mucoadhesiveness is a proposed strategy to increase the bioavailability of topical formulations. Herein, the design and optimization of MEL-loaded lipid-polymer hybrid nanoparticles (mel-LPHNs) using Design of Experiment (DoE) was performed. LPHNs consisted of PLGA-PEG polymer nanoparticles coated with a cationic lipid-shell. The optimized nanomedicine showed suitable size for ophthalmic administration (189.4 nm; PDI 0.260) with a positive surface charge (+39.8 mV), high encapsulation efficiency (79.8 %), suitable pH and osmolarity values, good mucoadhesive properties and a controlled release profile. Differential Scanning Calorimetry and Fourier-Transform Infrared Spectroscopy confirmed the encapsulation of melatonin in the systems and the interaction between lipids and polymer matrix. Biological evaluation in an in vitro model of diabetic retinopathy demonstrated enhanced neuroprotective and antioxidant activities of mel-LPHNs, compared to melatonin aqueous solution at the same concentration (0.1 and 1 μM). A modified Draize test was performed to assess the ocular tolerability of the formulation showing no signs of irritation. To the best our knowledge, this study reported for the first time the development of mel-LPHNs, a novel and safe hybrid platform suitable for the topical management of retinal diseases.