Intracellular sites of AQP2 S256 phosphorylation identified using inhibitors of the AQP2 recycling itinerary.

Vasopressin (VP)-regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the COOH-terminus; among them, serine 256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of this serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and Western blot analysis with phospho-specific antibodies. Using methyl-ß-cyclodextrin, cold block or bafilomycin, and taxol, we blocked AQP2 at the plasma membrane, in the perinuclear trans-Golgi network, and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared with baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the trans-Golgi network, or in cytoplasmic vesicles and that this event is dependent on the expression of PKA in these cells.NEW & NOTEWORTHY Phosphorylation of aquaporin-2 by PKA at serine 256 (S256) occurs in various subcellular locations during its recycling itinerary, suggesting that the protein complex necessary for AQP2 S256 phosphorylation is present in these different recycling stations. Furthermore, we showed, using PKA-null cells, that PKA activity is required for vasopressin-induced AQP2 phosphorylation. Our data reveal a complex spatial pattern of intracellular AQP2 phosphorylation at S256, shedding new light on the role of phosphorylation in AQP2 membrane accumulation.

NEI-01-Induced Arginine Deprivation Has Potent Activity Against Acute Myeloid Leukemia Cells Both In Vitro and In Vivo.

Recent studies have revealed that targeting amino acid metabolic enzymes is a promising strategy in cancer therapy. Acute myeloid leukemia (AML) downregulates the expression of argininosuccinate synthase (ASS1), a recognized rate-limiting enzyme for arginine synthesis, and yet displays a critical dependence on extracellular arginine for survival and proliferation. This dependence on extracellular arginine, also known as arginine auxotrophy, suggests that arginine deprivation would be a treatment strategy for AML. NEI-01, a novel arginine-depleting enzyme, is capable of binding to serum albumin to extend its circulating half-life, leading to a potent anticancer activity. Here we reported the preclinical activity of NEI-01 in arginine auxotrophic AMLs. NEI-01 efficiently depleted arginine both in vitro and in vivo NEI-01-induced arginine deprivation was cytotoxic to arginine auxotrophic AML cells through induction of cell-cycle arrest and apoptosis. Furthermore, the potent anti-leukemia activities of NEI-01 were observed in three different types of mouse models including human cell line-derived xenograft, mouse cell line-derived homografts in syngeneic mice and patient-derived xenograft. This preclinical data provide strong evidence to support the potential use of NEI-01 as a therapeutic approach in AML treatment.

Actin-related protein 2/3 complex plays a critical role in the aquaporin-2 exocytotic pathway.

The trafficking of proteins such as aquaporin-2 (AQP2) in the exocytotic pathway requires an active actin cytoskeleton network, but the mechanism is incompletely understood. Here, we show that the actin-related protein (Arp)2/3 complex, a key factor in actin filament branching and polymerization, is involved in the shuttling of AQP2 between the trans-Golgi network (TGN) and the plasma membrane. Arp2/3 inhibition (using CK-666) or siRNA knockdown blocks vasopressin-induced AQP2 membrane accumulation and induces the formation of distinct AQP2 perinuclear patches positive for markers of TGN-derived clathrin-coated vesicles. After a 20°C cold block, AQP2 formed perinuclear patches due to continuous endocytosis coupled with inhibition of exit from TGN-associated vesicles. Upon rewarming, AQP2 normally leaves the TGN and redistributes into the cytoplasm, entering the exocytotic pathway. Inhibition of Arp2/3 blocked this process and trapped AQP2 in clathrin-positive vesicles. Taken together, these results suggest that Arp2/3 is essential for AQP2 trafficking, specifically for its delivery into the post-TGN exocytotic pathway to the plasma membrane.NEW & NOTEWORTHY Aquaporin-2 (AQP2) undergoes constitutive recycling between the cytoplasm and plasma membrane, with an intricate balance between endocytosis and exocytosis. By inhibiting the actin-related protein (Arp)2/3 complex, we prevented AQP2 from entering the exocytotic pathway at the post-trans-Golgi network level and blocked AQP2 membrane accumulation. Arp2/3 inhibition, therefore, enables us to separate and target the exocytotic process, while not affecting endocytosis, thus allowing us to envisage strategies to modulate AQP2 trafficking and treat water balance disorders.

Hospital-in-the-Home experience of first 23 COVID-19 patients at a regional NSW hospital.

Ambulatory care is an important service for patients with the COVID-19 infection especially in a regional area where most of the patients underwent home isolation. Escalation of treatment and timely transition to inpatient care are critical when COVID-19 patients deteriorate. Equally important is ensuring transfer into facility is carried out in a well-planned, safe manner to prevent exposure to health care professionals as well as other inpatients. This study is a summary of our COVID Hospital-in-the-Home (HITH) service and clinical presentation of COVID-19 patients.

A modified arginine-depleting enzyme NEI-01 inhibits growth of pancreatic cancer cells.

Arginine deprivation cancer therapy targets certain types of malignancies with positive result in many studies and clinical trials. NEI-01 was designed as a novel arginine-depleting enzyme comprising an albumin binding domain capable of binding to human serum albumin to lengthen its half-life. In the present work, NEI-01 is shown to bind to serum albumin from various species, including mice, rat and human. Single intraperitoneal administration of NEI-01 to mice reduced plasma arginine to undetectable level for at least 9 days. Treatment of NEI-01 specifically inhibited cell viability of MIA PaCa-2 and PANC-1 cancer cell lines, which were ASS1 negative. Using a human pancreatic mouse xenograft model, NEI-01 treatment significantly reduced tumor volume and weight. Our data provides proof of principle for a cancer treatment strategy using NEI-01.

Validity of a tool designed to assess the preventability of potentially preventable hospitalizations for chronic conditions.

BACKGROUND: Potentially preventable hospitalizations (PPH) are defined as unplanned hospital admissions which could potentially have been prevented with the provision of effective, timely outpatient care. To better understand and ultimately reduce rates of PPH, a means of identifying those which are actually preventable is required. The Preventability Assessment Tool (PAT) was designed for use by hospital clinicians to assess the preventability of unplanned admissions for chronic conditions. OBJECTIVE: The present study examined the ability of the PAT to distinguish between those unplanned admissions which are preventable and those which are not, compared to the assessments of an Expert Panel. METHODS: Data were collected between November 2014 and June 2017 at three hospitals in NSW, Australia. Participants were community-dwelling patients with unplanned hospital admissions for congestive heart failure, chronic obstructive pulmonary disease, diabetes complications or angina pectoris. A nurse and a doctor caring for the patient made assessments of the preventability of the admission using the PAT. Expert Panels made assessments of the preventability of each admission based on a comprehensive case report and consensus process. RESULTS: There was little concordance between the hospital doctors and nurses regarding the preventability of admissions, nor between the assessments of the Expert Panel and the hospital nurse or the Expert Panel and the hospital doctor. CONCLUSIONS: The PAT demonstrated poor concurrent validity and is not a valid tool for assessing the preventability of unplanned hospital admissions. The use of Expert Panels provides a more rigorous approach to assessing the preventability of such admissions.

Discrimination of surface wear on obsidian tools using LSCM and RelA: pilot study results (area-scale analysis of obsidian tool surfaces).

This pilot study tests the reliability of laser scanning confocal microscopy (LSCM) to quantitatively measure wear on experimental obsidian tools. To our knowledge, this is the first use of confocal microscopy to study wear on stone flakes made from an amorphous silicate like obsidian. Three-dimensional surface roughness or texture area scans on three obsidian flakes used on different contact materials (hide, shell, wood) were documented using the LSCM to determine whether the worn surfaces could be discriminated using area-scale analysis, specifically relative area (RelA). When coupled with the F-test, this scale-sensitive fractal analysis could not only discriminate the used from unused surfaces on individual tools, but was also capable of discriminating the wear histories of tools used on different contact materials. Results indicate that such discriminations occur at different scales. Confidence levels for the discriminations at different scales were established using the F-test (mean square ratios or MSRs). In instances where discrimination of surface roughness or texture was not possible above the established confidence level based on MSRs, photomicrographs and RelA assisted in hypothesizing why this was so.

Effect of unilateral endobronchial valve insertion on pulmonary ventilation and perfusion: a pilot study.

BACKGROUND AND OBJECTIVE: The effects of bronchoscopic lung volume reduction (BLVR) on pulmonary ventilation and perfusion are incompletely understood. In this pilot trial, we investigated serial changes in regional ventilation and perfusion following unilateral endobronchial valve placement in COPD patients with heterogeneous emphysema. METHODS: At baseline and at days 30 and 90 following BLVR, subjects underwent lung function, 6MWD and St George's Respiratory Questionnaire. Ventilation and perfusion scintigraphy were performed to quantitate and serially compare regional differences. RESULTS: Six out of eight subjects completed the study; all had endobronchial valves targeting their left upper lobe. At day 90 post-BLVR, there was a trend towards an increase in FEV(1) and a mean reduction in St George's Respiratory Questionnaire score of nine units. In the targeted left upper zone there was reduced ventilation and perfusion. Ventilation and perfusion to the right lung; and specifically the right lower zone, significantly increased. CONCLUSIONS: There appears to be redistribution of ventilation and perfusion to the contralateral lung following endobronchial valve placement. This may be of importance when assessing patients for unilateral BLVR. Selecting patients with heterogeneous disease is emphasized, taking into consideration not just comparison between upper and lower lobes, but between left and right lungs. A larger trial is currently underway, guided by these findings.

Glossopharyngeal insufflation causes lung injury in trained breath-hold divers.

BACKGROUND AND OBJECTIVE: Glossopharyngeal insufflation (GI) is a technique practised by competitive breath-hold divers to enhance their performance. Using the oropharyngeal musculature, air is pumped into the lungs to increase the lung volume above physiological TLC. Experienced breath-hold divers can increase their lung volumes by up to 3 L. Although the potential for lung injury is evident, there is limited information available. The aim of this study was to examine whether there is any evidence of lung injury following GI, independent of diving. METHODS: Six male, competitive breath-hold divers were studied. CT of the thorax was performed during breath-holding at supramaximal lung volumes following GI (CT(GI)), and subsequently at baseline TLC (CT(TLC)). CT scans were performed a minimum of 3 days apart. Images were analysed for evidence of pneumomediastinum or pneumothorax by investigators who were blinded to the procedure. RESULTS: None of the subjects showed symptoms or signs of pneumomediastinum. However, in five of six subjects a pneumomediastinum was detected during the CT(GI). In three subjects a pneumomediastinum was detected on the CT(GI), but had resolved by the time of the CT(TLC). In two subjects a pneumomediastinum was seen on both the CT(GI) and the CT(TLC), and these were larger on the day that a maximal GI manoeuvre had been performed. The single subject, in whom a pneumomediastinum was not detected, was demonstrated separately to not be proficient at GI. CONCLUSIONS: Barotrauma was observed in breath-hold divers who increased their lung volumes by GI. The long-term effects of this barotrauma are uncertain and longitudinal studies are required to assess cumulative lung damage.

Spontaneous pneumomediastinum.

A male furniture removalist, 23 years of age, presented to his local doctor with neck swelling. He had an upper respiratory tract infection 2 weeks previously and was prescribed a course of oral antibiotics. He had returned to work 3 days before his presentation and described an episode of sudden onset, sharp, central chest pain while lifting a heavy object. The chest pain subsided within hours but he noticed a neck swelling at the onset of the pain, which has persisted. He pointed to a visible swelling on both sides of the posterior triangle of his neck. The swelling has persisted unchanged for the past 3 days. He smokes but has no other significant past medical history.

Intellectual functioning in presurgical patients with hypothalamic hamartoma and refractory epilepsy.

OBJECTIVE: The goal of the work described here was to examine the relationship between intellectual test performance in patients with hypothalamic hamartoma (HH) with refractory epilepsy and their seizure histories, as well as the size and neuroradiographic anatomical features of the HH. It was predicted that the level of estimated intelligence and the pattern of intellectual test performance would significantly correlate with the size of the HH and neuroanatomical features. METHOD: In this cross-sectional design study, 49 patients with HH between the ages of 5 and 55 years were classified by age at time of examination, as well as pattern of performance on the Wechsler intelligence scales. All patients were included in data analysis irrespective of their ability to participate in psychometric testing. Patients with a prior history of neurosurgical treatment were excluded. RESULTS: For those patients functionally capable of participating in cognitive testing (n=42), a summary index score, which estimated level of intellectual function (composed of the Vocabulary, Block Design, and Coding subtests of the Wechsler intelligence scales), was significantly correlated only with number of antiepileptic drugs (AEDs) the patient was taking at the time of evaluation (r=-0.66, n=38, P=0.05). In contrast, a categorization method addressing the pattern of intellectual test performance (including those patients who were not functionally capable of participating in cognitive testing, n=49) was significantly correlated with number of AEDs (r=+0.35, n=48, P=0.01), size of HH (r=+0.38, n=49, P=0.01), presence of precocious puberty (PP: r=+0.41, n=49, P=0.01), and anatomical classification of HH (r=+0.39, n=49, P=0.01). CONCLUSIONS: The findings confirm the wide range of cognitive functioning in the population of patients with HH and refractory epilepsy, and suggest that multiple variables are correlated with intellectual test performance in patients with HH with refractory epilepsy. Although the present cross-sectional design study does not answer the question of whether or not epilepsy severity produces lower intelligence in this patient population, number of AEDs and neuroanatomical features of the HH lesion are identified as being significantly related to cognitive performance in this patient sample.

The histopathology of hypothalamic hamartomas: study of 57 cases.

Hypothalamic hamartomas (HHs) are rare developmental tumors that cause seizures or pituitary axis dysfunction, usually beginning in childhood. We analyzed HH tissue from 57 patients whose tumors were resected through recently developed transcallosal interforniceal and transventricular endoscopic surgical approaches. All cases were composed of abnormally distributed but cytologically normal neurons and glia, including fibrillary astrocytes and oligodendrocytes. Neuronal elements predominated in most cases, but a relative increase in astrocytic elements was seen with increasing age. All had various sized nodular foci of neurons as well as areas of diffusely distributed neurons with interspersed glial cells. Smaller neurons predominated, and most cases had only a few interspersed large ganglion cells. Immunohistochemistry demonstrated extensive production of synapse-associated proteins. Immunohistochemistry for phosphorylated and nonphosphorylated neurofilament and alpha-internexin demonstrated staining patterns consistent with mature neurons. In contrast to cortical dysplasia, atypical large ganglion-like balloon cells were almost never seen. In summary, although their number and distribution vary, mature smaller neurons were the most prominent and most consistent histologic feature of HH. Nodules of these small neurons were a universal feature of the microarchitecture of HH lesions associated with epilepsy. Characterization of these neurons may aid in understanding the mechanism of seizure development in HH.

The hypothalamic hamartoma: a model of subcortical epileptogenesis and encephalopathy.

Although uncommon, the hypothalamic hamartoma (HH) is often associated with a devastating clinical syndrome, which may include refractory epilepsy, progressive cognitive decline, and deterioration in behavioral and psychiatric functioning. Contrary to conventional thinking which attributed seizure origin to cortical structures, the hamartoma itself has now been firmly established as the site of intrinsic epileptogenesis for the gelastic seizures (i.e., characterized by unusual mirth) peculiar to this disorder. It also appears that the HH contributes to a process of secondary epileptogenesis, with eventual cortical seizure onset of multiple types in some patients. Anticonvulsant medications are known to be poorly effective in this disorder. Treatment, including some innovative approaches to surgical resection, is now targeted directly at the HH itself, with impressive results. Younger patients, in particular, may avoid the deteriorating course described earlier. Access to tissue from larger numbers of patients at single or collaborating centers specializing in HH surgery will allow for research into the fundamental mechanisms producing this little understood disorder. Refractory epilepsy associated with HH is the premier human model for subcortical epilepsy and an excellent model for secondary epileptogenesis and epileptic encephalopathy.

Acyclic Stereoselection in the Ortho Ester Claisen Rearrangement.

The ortho ester Claisen rearrangement of trisubstituted allylic alcohols exhibits significant levels of diastereoselection. In E allylic alcohols, a 1,3-diaxial interaction develops in the chairlike transition state leading to the anti isomer, rendering the reaction syn selective by a factor of 3-5 to 1. In Z allylic alcohols, the 1,3-diaxial interaction develops in the transition state leading to the syn isomer, generating an anti:syn selectivity of 6-15 to 1. The relative stereochemistry of the syn isomer was confirmed independently by the synthesis of the mycotoxin botryodiplodin.