The Role of Music Therapy for Children Undergoing Cancer Treatment in Singapore.

The aim of this study was to explore the benefits of music therapy (MT) for children with cancer over the course of their treatment in an acute paediatric hospital setting in Singapore. Twenty-five children undergoing cancer treatment received MT sessions as part of a multidisciplinary team rehabilitation intervention from March 2017 to January 2020. A total of 37 individualised goals were developed by the music therapist for the cohort. Goals were scored via the Goal Attainment Scale at 3-month intervals up to 1 year. Descriptive statistics and correlation analysis were used to evaluate the findings. The rate of goal achievement was 89.2% over 180 MT sessions (M = 7.20, SD = 6.45). Children diagnosed with brain tumours had the highest frequency of MT sessions (M = 9.11, SD = 7.79). Most of the goals targeted the regulation of mood and morale through music. There was a positive correlation found between goals and sessions (rs = 0.56, p = 0.004). Age of the children was not correlated with the number of sessions received (rs= -0.19, p = 0.354). MT has been found to be an accessible and effective intervention in addressing functional and emotional goals for children across all ages who are undergoing cancer treatment.

A Meta-Analytic Review of the Efficacy of Physical Exercise Interventions on Cognition in Individuals with Autism Spectrum Disorder and ADHD.

This review evaluates the efficacy of using physical exercise interventions on improving cognitive functions in individuals with autism spectrum disorder (ASD) and/or attention deficit hyperactivity disorder (ADHD). This review includes a meta-analysis based on a random-effects model of data reported in 22 studies with 579 participants aged 3-25 year old. The results revealed an overall small to medium effect of exercise on cognition, supporting the efficacy of exercise interventions in enhancing certain aspects of cognitive performance in individuals with ASD and/or ADHD. Specifically, similar to the general population literature, the cognitive benefits of exercise are not consistent across all aspects of cognitive functions (i.e., some areas are not improved). The clinical significance of the reported effect sizes is also considered.

Recruitment of coat-protein-complex proteins on to phagosomal membranes is regulated by a brefeldin A-sensitive ADP-ribosylation factor.

Particle internalization in macrophages is followed by a complex maturation process. We have previously observed that proteins bound to phagocytosed particles are sorted from phagosomes into a heterogeneous population of vesicles that fuse with endosomes. However, the mechanism and the protein machinery involved in the formation of these phagosome-derived vesicles are largely unknown. It has been shown that vesicles coated with coat protein complex type I (COPI) have a role in both secretion and endocytosis. To address the possibility that COPI proteins might participate in the formation of phagosome-derived vesicles we studied the recruitment of beta-COP to highly purified phagosomes. The binding of beta-COP to phagosomal membranes was regulated by nucleotides and inhibited by brefeldin A (BFA). An ADP-ribosylation factor 1 (ARF1) mutant defective in GTP hydrolysis supported the binding of beta-COP to phagosomes independently of added nucleotide. AlF(4) and Gbetagamma subunits, agents known to modulate heterotrimeric G-protein activity, were tested in the beta-COP binding assay. AlF(4) increased beta-COP association, whereas binding was inhibited by the addition of Gbetagamma subunits. Our results suggest that COP proteins are recruited to phagosomal membranes by a mechanism that involves heterotrimeric GTP-binding proteins and a BFA-sensitive ARF. In addition, our findings indicate that COPI proteins are involved in the recycling of components from phagosomes to the cell surface.

Recovery of distal nephron enzyme activity after release of unilateral ureteral obstruction.

PURPOSE: Unilateral ureteral obstruction (UUO) for 24 hours results in a severe compromise of distal tubular function. The acidification defect is believed to be localized in the collecting duct. To characterize distal tubular function recovery one month after junction release, clearance studies in whole animals and enzyme studies in microdissected segments were performed in an experimental model of unilateral ureteral obstruction. MATERIALS AND METHODS: Following release of ureteral obstruction of 24 hours duration, a significant decrease of whole kidney glomerular filtration rate was observed in the postobstructed kidney (POK) with a marked increase in urinary pH, fractional excretion of bicarbonate (FEHCO3-) and decrease in urinary osmolality. By orthograde stop flow experiment, bicarbonate excretion rate (Fr:Ff HCO3-/Fr:Ff Inutest) increased in the first and second urine fractions of 120 microl. corresponding to the collecting segment in the POK, one day after release. Decrease in U-P pCO2 (p<0.01) suggested an impaired H+ secretion on distal nephron in POK. Recovery of inulin clearance and values of urinary pH, FEHCO3- and urinary osmolality near contralateral and control kidneys were observed thirty days following ureteral release. The decline in enzyme activity in the distal nephron due to structural damage from high intratubular pressure was evaluated. Bafilomycin sensitive H+ -ATPase activity measurement in the medullary collecting duct segments of the POK showed an important decrease (68%), with lightly reduced activity (20%) in the cortical collecting duct, 24 hours after obstruction release. Localized in the connecting tubule cells and secreted into the tubular fluid in the late distal nephron, renal kallikrein has been involved in bicarbonate transport at cortical collecting duct segments. The renal kallikrein-like activity was reduced in POK (p<0.01). RESULTS: Recovery of enzyme activity was shown thirty days after unilateral ureteral obstruction. Our results show severe functional damage of the collecting duct after 24 hours of unilateral ureteral obstruction. H+ -ATPase activity was markedly decreased on medullary collecting duct segments. CONCLUSIONS: A correlation between the functional impairment of distal H+ secretion and decreased distal nephron enzyme activity has been shown. Recovery of both the functional and the enzyme activity at the distal nephron was demonstrated thirty days after obstruction release.

Reconstitution of phagosome-lysosome fusion in streptolysin O-permeabilized cells.

We have reconstituted fusion between phagosomes and lysosomes in streptolysin O-permeabilized J774-E macrophages. Fusion was assessed by measuring the delivery of avidin-conjugated horseradish peroxidase pre-internalized into lysosomes to phagosomes containing biotinylated beta-glucuronidase-conjugated paramagnetic beads (1-2 microm). Fusion was dependent on energy and exogenously supplied cytosol. Phagosome-lysosome fusion was greatly inhibited when microtubules were depolymerized by nocodazole treatment, suggesting that fusion occurs via microtubule-dependent transport. Furthermore, fusion was inhibited by GTPgammaS and Rab GDP dissociation inhibitor. These results suggest that rab proteins are involved in the regulation of fusion. Lastly, anti-NEM-sensitive factor (NSF) antibodies inhibited fusion, and addition of recombinant NSF wild type partially restored the fusogenic activity, indicating that NSF is required for fusion between phagosomes and lysosomes.

Calmodulin regulates endosome fusion.

Calmodulin (CaM) has previously been implicated in regulated exocytosis, transcytosis, and receptor recycling. We have investigated the role of CaM in endocytic transport by examining the effects of several CaM antagonists in intact cells. We present evidence indicating that the mixing of sequentially internalized ligands is inhibited by CaM antagonists, indicating that CaM may play a general role in regulating endosomal membrane trafficking. To address the specific events that are affected by CaM we studied its role in an in vitro assay that reconstitutes fusion among endosomes. CaM antagonists inhibited endosome fusion, and the inhibition was reversed by the addition of CaM. Moreover, we found that Ca2+ stimulates fusion among endosomes and that addition of CaM stimulates fusion beyond that produced by Ca2+ alone. Our data indicate that one of the possible targets for CaM in endosome fusion is the CaM-dependent kinase II. We propose that CaM regulates endocytic transport by modulating an essential component(s) of the membrane traffic machinery.

Phagocytosed live Listeria monocytogenes influences Rab5-regulated in vitro phagosome-endosome fusion.

Survival or destruction of a pathogen following phagocytosis depends, in part, on fusion events between the phagosome and the endosomal or lysosomal compartments. Here we use an in vitro assay to show that phagosome-endosome fusion is regulated by the small GTPase rab5 and that fusion events are influenced by an internalized live organism, Listeria monocytogenes (LM). We compare the in vitro fusion of phagosomes containing heat-killed organisms (dead LM) with that of phagosomes containing a live nonhemolytic mutant (live LMhly-). Unlike the wild-type organism, LMhly- remains trapped inside the phagosome. Phagosome-endosome fusion was reconstituted using biotinylated organisms and endosomes containing horseradish peroxidase conjugated with avidin. With both live LMhly- and dead LM preparations, in vitro phagosome-endosome fusion was time-, temperature-, and cytosol-dependent. Live LMhly- phagosomes exhibited a faster rate of fusion. Fusion in both preparations was regulated by rab5 and possibly by other GTPases. Anti-rab5 antibodies and immunodepletion of cytosolic rab5 inhibited fusion. Addition of glutatione S-transferase-rab5 in the GTP form stimulated phagosome-endosome fusion, whereas addition of a dominant negative mutant of rab5 blocked fusion. Purified live LMhly- phagosomal membranes were enriched in rab5 as revealed by Western blotting, compared with dead LM phagosomes. Fusion of endosomes with dead LM containing phagosomes required ATP and was inhibited by ATP depletion and by N-ethylmaleimide (NEM) and anti-NEM-sensitive factor (NSF) antibodies. Unexpectedly, phagosome-endosome fusion with live LMhly--containing phagosomes was not inhibited by ATP depletion nor by NEM or anti-NSF antibodies. Western blot analysis revealed that live LMhly--containing phagosomes were enriched for membrane-bound NSF, while dead LM containing phagosomes contained low or undetectable quantities. Washing live LMhly--containing phagosomes with 0.5 M KCl removed NSF associated with the membranes and rendered them NEM, ATP, anti-NSF antibody sensitive for fusion. We conclude that rab5 regulates phagosome-endosome fusion and that live microorganisms can up-regulate this process by recruiting rab5 to the membrane.

Tubulin aggregates induced by Ni2+ present microtubular characteristics.

Rat brain tubulin in a proper buffered solution became insoluble in the presence of 10 mM NiCl2, and sedimented at centrifugal forces as low as 500 x g for 30 min. Both nickel-sedimented and microtubular tubulin conserved 65% of colchicine binding activity after 25 days of storage at -20 degrees C. However in brain cytosol, only 9% of the initial binding activity was conserved. The electrophoretic mobility of tubulin recovered from aggregates also remained unaltered. Therefore the aggregates formed with Ni2+ share important physicochemical properties with microtubules.

Tubulin aggregates induced by Ni2+ present microtubular characteristics

Rat brain tubulin in a proper buffered solution became insoluble in the presence of 10 mM NiCl2, and sedimented at centrifugal forces as low as 500 x g for 30 min. Both nickel-sedimented and microtubular tubulin conserved 65 of colchicine binding activity after 25 days of storage at -20 degrees C. However in brain cytosol, only 9 of the initial binding activity was conserved. The electrophoretic mobility of tubulin recovered from aggregates also remained unaltered. Therefore the aggregates formed with Ni2+ share important physicochemical properties with microtubules.

Tubulin aggregates induced by Ni2+ present microtubular characteristics

Rat brain tubulin in a proper buffered solution became insoluble in the presence of 10 mM NiCl2, and sedimented at centrifugal forces as low as 500 x g for 30 min. Both nickel-sedimented and microtubular tubulin conserved 65 of colchicine binding activity after 25 days of storage at -20 degrees C. However in brain cytosol, only 9 of the initial binding activity was conserved. The electrophoretic mobility of tubulin recovered from aggregates also remained unaltered. Therefore the aggregates formed with Ni2+ share important physicochemical properties with microtubules.(AU)

Heterotrimeric G proteins interact with the small GTPase ARF. Possibilities for the regulation of vesicular traffic.

Trimeric G proteins have emerged as important regulators of membrane trafficking. To explore a role for G beta gamma in endosome fusion, we have taken advantage of beta-adrenergic receptor kinase (beta ARK), an enzyme translocated to membranes by interaction with G beta gamma. The COOH terminus of beta ARK (beta ARKct) has a G beta gamma-binding domain which blocks some G beta gamma-mediated processes. We found that beta ARKct and peptide G, a peptide derived from beta ARKct, inhibit in vitro endosome fusion. Interestingly, peptide G and ARF share sequence similarity. Peptide G and beta ARKct reversed ARF-mediated inhibition of endosome fusion and blocked ARF binding to membranes. Using an ARF fusion protein, we show that both G beta gamma and G alpha s interact with the small GTPase ARF, an interaction that is regulated by nucleotide binding. We conclude that G proteins may participate in the regulation of vesicular trafficking by directly interacting with ARF, a cytosolic factor required for transport.

Microtubules mediate cellular 25-hydroxyvitamin D3 trafficking and the genomic response to 1,25-dihydroxyvitamin D3 in normal human monocytes.

The genomic actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the intracellular vitamin D receptor (VDR). Although immunocytochemistry has shown that disruption of microtubular assembly prevents nuclear access of the sterol-VDR complex, the role of microtubules in the response to 1,25(OH)2D3 has not been studied in viable cells. Our studies examined this interaction in normal human monocytes. Monocytes convert 25(OH)D3 to 1,25(OH)2D3 and to 24-hydroxylated metabolites more polar than 1,25(OH)2D3. Microtubule disruption totally abolished the ability of exogenous 1,25(OH)2D3 to suppress its own synthesis and to induce 24-hydroxylase mRNA and activity, without affecting either total 1,25(OH)2D3 uptake or maximal 1,25(OH)2D3-VDR binding. Thus, intact microtubules are essential for 1,25(OH)2D3-dependent modulation of gene transcription. Interestingly, microtubule disruption also decreased monocyte 1,25(OH)2D3 synthesis, not by decreasing the Vmax of monocyte mitochondrial 1 alpha-hydroxylase but through an increase in the Km for 25(OH)2D3. We examined 25(OH)D3 transport. Microtubule disruption did not affect total cellular 25(OH)D3 uptake but reduced its intracellular trafficking to the mitochondria. Thus, microtubules participate in intracellular 25(OH)D3 transport, and their integrity determines normal 1,25(OH)2D3 synthesis.

In vitro reconstitution of phagosome-endosome fusion: evidence for regulation by heterotrimeric GTPases.

We have assessed the role of heterotrimeric GTPases on in vitro fusion of phagosomes and endosomes. Highly purified phagosomes were found to contain G alpha s, G alpha i1, G alpha i2, G alpha i3, and G beta subunits of heterotrimeric GTP-binding proteins. A functional role for G proteins was established using an in vitro phagosome-endosome fusion assay. First, addition of AlF4- and purified G beta gamma subunits to the in vitro assay blocked fusion, indicating that heterotrimeric G proteins may play a role, either direct or indirect, in phagosome maturation. Second, a striking inhibitory effect was observed when the vesicles were incubated with peptides that preferentially activate G alpha s. A similar effect on phagosome-endosome fusion was observed with cholera toxin, a reagent known to activate G alpha s. Our results suggest that one or more heterotrimeric G proteins, including Gs, mediate and/or regulate phagosome-endosome fusion.

Membrane trafficking along the phagocytic pathway.

Phagosome maturation involves extensive remodelling of the phagosomal membrane as a result of intracellular transport events. Newly formed phagosomes exchange membrane-associated and soluble proteins with early endosomes by fusion. Budding of vesicles from the phagosome and fusion with Golgi-derived vesicles may also contribute to the remodelling of the phagosomal compartment. As a consequence of changes in membrane composition, phagosomes acquire the ability to fuse with late endocytic compartments. In vitro reconstitution and other studies suggest that the trafficking events underlying phagosome maturation require several GTP-binding proteins, including Rab5 and Galphas', NSF-SNAP-SNARE complexes and coatomers.

Tubulin aggregates induced by Ni2+ present microtubular characteristics.

Rat brain tubulin in a proper buffered solution became insoluble in the presence of 10 mM NiCl2, and sedimented at centrifugal forces as low as 500 x g for 30 min. Both nickel-sedimented and microtubular tubulin conserved 65

of colchicine binding activity after 25 days of storage at -20 degrees C. However in brain cytosol, only 9

of the initial binding activity was conserved. The electrophoretic mobility of tubulin recovered from aggregates also remained unaltered. Therefore the aggregates formed with Ni2+ share important physicochemical properties with microtubules.

Calcium-dependent fusion among endosomes.

Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we have previously reported that, in the absence of externally added calcium, endosome fusion requires cytosol, ATP, and is sensitive to N-ethylmaleimide (NEM) and to anti-NEM-sensitive factor (NSF) antibody. This cytosol-dependent fusion is regulated by monomeric and heterotrimeric GTP-binding proteins. Further studies have revealed, however, that in the presence of micromolar concentrations of free calcium, fusion is observed even in the absence of cytosol and ATP. At the electron microscope level, Ca(2+)-dependent endosome aggregation and fusion were similar to that observed for cytosol-dependent fusion. Calcium-dependent fusion was not affected by non-hydrolyzable analogs of GTP or GDP nor by NEM or anti-NSF antibody. However, Ca(2+)-dependent fusion was abrogated by trypsin treatment of the vesicles or by a membrane wash with 60 mM EDTA indicating that peripheral proteins are required. An anti-annexin II antibody and an annexin II peptide blocked Ca(2+)-dependent fusion by 50%. After the EDTA wash, Ca(2+)-dependent fusion was reconstituted by addition of purified annexin II and arachidonic acid. We conclude that endosomes can fuse by two mechanisms, one that has an absolute requirement for calcium and is probably mediated by annexins, and another that does not require calcium.

Inhibition of endocytic transport by aluminum fluoride implicates GTPases as regulators of endocytosis.

It is now well established that GTP-binding proteins are important regulators of vesicular transport. Recent work has shown that multiple GTPases (both monomeric and heterotrimeric) are required for trafficking. In the present study we have used aluminum fluoride (AIF), a reagent that activates trimeric G proteins, as a tool to study the involvement of this family of GTPases in the regulation of endocytosis in intact cells. Our results indicate that AIF inhibits fusion of early endosomes with an intracellular proteolytic compartment. Using the mixing of sequentially internalized ligands as a measure of endocytosis, we found that AIF inhibited endocytic transport as assessed by both biochemical and morphological methods. Taken together these results suggest that AIF affects membrane fusion, a common step in vesicular transport. To further examine the effects of AIF we tested this compound in a cell-free assay that reconstitutes fusion among endosomes. AIF affected endosomal fusion in a different way than did GTP gamma S, an agent that activates both trimeric and small GTPases. Our results suggest that the coordinated activation of both classes of GTPases are required for efficient endocytic transport.

Colchicine binding to tubulin from brain homogenates in the presence of sugars, glycols and metal ions. Effect of nickel ions on the tubulin solubility.

The fact that glycerol preserves microtubules from depolymerizing in vitro, and that some ions such as Ca(II) and Mg(II), regulate the assembly-disassembly process of these structures, induced us to study the effect of several sugars, glycols and metal ions on solubility and colchicine affinity of tubulin in rat brain homogenates, and of purified microtubular protein. Inhibition of colchicine binding was significant with glycerol, polyethylene glycol 1000 (PEG-2) and the ions Al(III), Co(II), Ni(II), while compounds structurally related to glycerol (glucose and sucrose) did not inhibit it. Mannitol, instead, increased the activity a 47% over control. Apparently the presence of some compounds in brain homogenates [PEG-2 (1000) and Ni(II)] favored tubulin sedimentation when these latter were centrifuged at 100,000 x g for 150 min at 20 degrees C, but the form in which tubulin becomes aggregated in the pellet is unknown. Nickel ion made insoluble microtubular protein of homogenates and the purified one by more than 90% without causing significant inhibition of the colchicine binding. The sediment containing nickel-treated two cycles purified microtubular protein observed with the electron microscope did not present microtubules, but it revealed the presence of irregular, wavy and stretched structures bearing highly dense dotted material. The sediments became soluble in phosphate-glutamate buffer (pH 6.8) and, when incubated in polymerizing conditions, gave rise to microtubules undistinguishable from those prepared with untreated purified protein.

Colchicine binding to tubulin from brain homogenates in the presence of sugars, glycols and metal ions. Effect of nickel ions on the tubulin solubility.

The fact that glycerol preserves microtubules from depolymerizing in vitro, and that some ions such as Ca(II) and Mg(II), regulate the assembly-disassembly process of these structures, induced us to study the effect of several sugars, glycols and metal ions on solubility and colchicine affinity of tubulin in rat brain homogenates, and of purified microtubular protein. Inhibition of colchicine binding was significant with glycerol, polyethylene glycol 1000 (PEG-2) and the ions Al(III), Co(II), Ni(II), while compounds structurally related to glycerol (glucose and sucrose) did not inhibit it. Mannitol, instead, increased the activity a 47

over control. Apparently the presence of some compounds in brain homogenates [PEG-2 (1000) and Ni(II)] favored tubulin sedimentation when these latter were centrifuged at 100,000 x g for 150 min at 20 degrees C, but the form in which tubulin becomes aggregated in the pellet is unknown. Nickel ion made insoluble microtubular protein of homogenates and the purified one by more than 90

without causing significant inhibition of the colchicine binding. The sediment containing nickel-treated two cycles purified microtubular protein observed with the electron microscope did not present microtubules, but it revealed the presence of irregular, wavy and stretched structures bearing highly dense dotted material. The sediments became soluble in phosphate-glutamate buffer (pH 6.8) and, when incubated in polymerizing conditions, gave rise to microtubules undistinguishable from those prepared with untreated purified protein.