The impact of COVID-19 on the well-being of Victorian children born with cleft lip and/or palate.

BACKGROUND: A longitudinal cohort study was established to investigate the well-being of children born with cleft lip and/or palate (CL/P) during the COVID-19 pandemic, in Victoria, Australia. MATERIALS AND METHODS: The Royal Children's Hospital cleft service database was used to identify children aged between 4 and 17 years old born with an isolated CL/P. Families of eligible children who consented to participate were asked to complete the Strengths and Difficulties Questionnaire (SDQ) between October and December 2020 and again 6-month later. SDQ results from typically developing Australian children during the COVID-19 pandemic were utilized from a previously published study. RESULTS: 63 parents completed the baseline questionnaire, with 44 completing the 6-month follow-up. For participants at baseline, the mean age was 8.9 years, with 55% male. All outcome domains of the SDQ improved between baseline and timepoint 2, with the difference in total difficulties scores being statistically significant, indicating a reduction in total difficulties at timepoint 2, associated with the easing of COVID-19 restrictions. When compared with the Australian population during the COVID-19 pandemic, Victorian children born with CL/P had lower SDQ scores for all difficulties outcome domains, with statistically significant results for conduct problems, hyperactivity, peer problems and total difficulties, indicating fewer difficulties for children born with CL/P. CONCLUSIONS: Children born with CL/P experienced fewer difficulties when compared with the typically developing Australian population during the COVID-19 pandemic. The level of restrictions imposed because of the pandemic also had little influence on the well-being of these children.

Characterization of large basking shark Cetorhinus maximus aggregations in the western North Atlantic Ocean.

Cetorhinus maximus aggregations recorded during extensive aerial survey efforts off the north-eastern United States between 1980 and 2013 included aggregations centring on sightings with group sizes of at least 30 individuals. These aggregations occurred in summer and autumn months and included aggregation sizes of up to 1398 individuals, the largest aggregation ever reported for this species. The aggregations were associated with sea surface temperatures of 13-24° C and chlorophyll-a concentrations of 0·4-2·6 mg m-3 and during one aggregation, a high abundance of zooplankton prey was present. Photogrammetric tools allowed for the estimation of total body lengths ranging between 4 and 8 m. Characterization of these events provides new insight into the potential biological function of large aggregations in this species.

Social function assessment tools for children and adolescents: a systematic review from 1988 to 2010.

Advances in neuroscience have added to the understanding of social functioning which has become an increasing area of focus in the psychology and neuropsychology literature. Given importance of appropriate social functioning to everyday interactions, as well as psychological well-being, accurately identifying and documenting such functions constitute a critical undertaking for both researchers and clinicians in psychology and related health professions. This review aimed to identify available social function assessment tools for children and adolescents using a comprehensive search method. Eighty-six measures were identified. Information on the assessment tools including the theoretical model they are based on, age range, sample used in development, and psychometric information are described. Results will aid researchers, psychologists and other health professionals in the selection of an appropriate tool to assess social function.

Anhydrobiosis in yeast: stabilization by exogenous lactose.

We have found that incubation in lactose solutions (0.75 M) of yeast culture Saccharomyces cerevisiae sensitive to dehydration damage increased the stability of the cells during dehydration. Simultaneously with this increase in viability, a decrease in plasma membrane permeability during rehydration was seen. Using Fourier transform infrared spectroscopy to measure lipid phase transitions, we observed that the lactose treatment depressed the membrane phospholipid phase transition temperature in a sensitive culture of dry yeast. As a result, it leads to the decrease in the damages of molecular organization of membranes during rehydration of dry yeast cells, thus reducing leakage from the cells.

The trehalose myth revisited: introduction to a symposium on stabilization of cells in the dry state.

This essay is an introduction to a series of papers arising from a symposium on stabilization of cells in the dry state. Nearly all of these investigations have utilized the sugar trehalose as a stabilizing molecule. Over the past two decades a myth has grown up about special properties of trehalose for stabilization of biomaterials. We review many of such uses here and show that under ideal conditions for drying and storage trehalose has few, if any, special properties. However, under suboptimal conditions trehalose has some distinct advantages and thus may remain the preferred excipient. We review the available mechanisms for introducing trehalose into the cytoplasm of living cells as an introduction to the papers that follow.

Membrane reorganization during chilling: implications for long-term stabilization of platelets.

This essay is a review of the various biophysical and biochemical events that make up the factors responsible for platelet cold-induced activation. It describes the formation of large membrane domains composed of raft aggregates that occur during chilling and storage. It also presents strong evidence that platelet membranes undergo lateral phase separation during prolonged storage in the cold and suggests that raft aggregation and lateral phase separation are key events which must be obviated to stabilize platelets and store them either in the frozen or in the dry state.

Comparison of the solution conformation and dynamics of antifreeze glycoproteins from Antarctic fish.

The (1)H- and (13)C-NMR spectra of antifreeze glycoprotein fractions 1-5 from Antarctic cod have been assigned, and the dynamics have been measured using (13)C relaxation at two temperatures. The chemical shifts and absence of non-sequential (1)H-(1)H NOEs are inconsistent with a folded, compact structure. (13)C relaxation measurements show that the protein has no significant long-range order, and that the local correlation times are adequately described by a random coil model. Hydroxyl protons of the sugar residues were observed at low temperature, and the presence of exchange-mediated ROEs to the sugar indicate extensive hydration. The conformational properties of AFGP1-5 are compared with those of the previously examined 14-mer analog AFGP8, which contains proline residues in place of some alanine residues (Lane, A. N., L. M. Hays, R. E. Feeney, L. M. Crowe, and J. H. Crowe. 1998. Protein Sci. 7:1555-1563). The infrared (IR) spectra of AFGP8 and AFGP1-5 in the amide I region are quite different. The presence of a wide distribution of backbone torsion angles in AFGP1-5 leads to a rich spectrum of frequencies in the IR spectrum, as interconversion among conformational states is slow on the IR frequency time scale. However, these transitions are fast on the NMR chemical shift time scales. The restricted motions for AFGP8 may imply a narrower distribution of possible o, psi angles, as is observed in the IR spectrum. This has significance for attempts to quantify secondary structures of proteins by IR in the presence of extensive loops.

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation.

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.

Are lipid phase transitions responsible for chilling damage in human platelets?

In previous studies we have proposed that the well-known chilling-induced activation of human blood platelets can be ascribed at least in part to a thermotropic phase transition in membrane lipids. The evidence that this is the case is reviewed and amplified in this review, followed by an examination of the available physical data concerning phase transitions in lipid mixtures that mimic the mixture found in platelet membranes. Assuming complete mixing at all temperatures and equal contributions of the members of the mixture to the phase transition, the lipid mixture found in platelets should give values for Tm ranging from about 1 degrees C to about 16 degrees C, depending on the isomers present in the mixture. (The former value is not in agreement with the observed Tm, but the latter is in excellent agreement.) However, examination of the phase diagram for a binary pair of lipids found in platelet membranes shows that ideal mixing almost certainly does not occur; instead of a linear phase diagram, a convex one was obtained. This shape for the phase diagram, which would displace Tm to an unexpectedly elevated temperature, is in agreement with previously published phase diagrams for mixtures of this type. The prediction, based on thermodynamic properties of lipids found in the platelets, is that Tm will be displaced upward in more complex mixtures of the composition found in platelets, a prediction that requires experimental testing.

Effect of sugars on headgroup mobility in freeze-dried dipalmitoylphosphatidylcholine bilayers: solid-state 31P NMR and FTIR studies.

The effect of the carbohydrates trehalose, glucose, and hydroxyethyl starch (HES) on the motional properties of the phosphate headgroup of freeze-dried dipalmitoylphosphatidylcholine (DPPC) liposomes was studied by means of 31P NMR, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The results show that trehalose, which is a strong glass former (Tg = 115 degreesC), elevates the onset of the lipid headgroup rotations and preserves some rotational mobility of the phosphate headgroups after cooling from the liquid-crystalline state. Glucose (Tg = 30 degreesC), a very effective depressant of the phase transition temperature of freeze-dried DPPC, markedly elevates the initiation of the temperature of headgroup rotations. On the other hand, the monosaccharide does not preserve the headgroup disordering when cooled from the liquid-crystalline state. These effects are consistent with formation of hydrogen bonds between the OH groups of the sugar and the polar headgroups of DPPC. They show, however, that hydrogen bonding is not sufficient for preservation of the dynamic properties of freeze-dried DPPC. HES, although a very good glass former (Tg > 110 degreesC), does not depress the phase transition temperature and affects only slightly the rotational properties of freeze-dried DPPC. This lack of effect of HES is associated with the absence of direct interactions with the lipid phosphates, as evidenced by the FTIR results. These data show that vitrification of the additive is not sufficient to affect the dynamic properties of dried DPPC.

Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from Antarctic cod.

The 1H and 13C NMR spectra of a 14-residue antifreeze glycopeptide from Antarctic cod (Tetramatomnus borchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 degrees C of 3-4 ns. The relaxation data and lack of long-range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16-residue analogue containing an Arg-Ala dipeptide added to the C-terminus. Molecular modeling also showed no evidence of long-range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or beta turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage.

Interactions of arbutin with dry and hydrated bilayers.

The glycosylated hydroquinone arbutin (4-hydroxyphenyl-beta-D-glucopyranoside) is abundant in certain resurrection plants, which can survive almost complete dehydration for prolonged periods. Little is known about the role of arbutin in vivo, but it is thought to contribute toward survival of the plants in the dry state. We have investigated the interactions of arbutin with model membranes under conditions of high and low hydration, as well as the possible participation of arbutin in carbohydrate glasses formed at low water contents. Retention of a trapped soluble marker inside large unilamellar vesicles and fusion of vesicles was monitored by fluorescence spectroscopy. Effects of arbutin on glass-transition temperatures and hydrated membrane phase-transition temperatures were measured by differential scanning calorimetry. The possible insertion of arbutin into membrane bilayers was estimated by following arbutin auto-fluorescence. Evidence is presented that arbutin does not change the glass-transition temperature of a sucrose/trehalose glass, but that arbutin does interact with hydrated membranes by insertion of the phenol moiety into the lipid bilayer. This interaction causes increased membrane leakage during air-drying by a mechanism other than vesicle-vesicle fusion. Implications of these effects on the dehydrated plant cells, as well as possible methods of obviating the damage, are discussed.

The role of vitrification in anhydrobiosis.

Numerous organisms are capable of surviving more or less complete dehydration. A common feature in their biochemistry is that they accumulate large amounts of disaccharides, the most common of which are sucrose and trehalose. Over the past 20 years, we have provided evidence that these sugars stabilize membranes and proteins in the dry state, most likely by hydrogen bonding to polar residues in the dry macromolecular assemblages. This direct interaction results in maintenance of dry proteins and membranes in a physical state similar to that seen in the presence of excess water. An alternative viewpoint has been proposed, based on the fact that both sucrose and trehalose form glasses in the dry state. It has been suggested that glass formation (vitrification) is in itself sufficient to stabilize dry biomaterials. In this review we present evidence that, although vitrification is indeed required, it is not in itself sufficient. Instead, both direct interaction and vitrification are required. Special properties have often been claimed for trehalose in this regard. In fact, trehalose has been shown by many workers to be remarkably (and sometimes uniquely) effective in stabilizing dry or frozen biomolecules, cells, and tissues. Others have not observed any such special properties. We review evidence here showing that trehalose has a remarkably high glass-transition temperature (Tg). It is not anomalous in this regard because it lies at the end of a continuum of sugars with increasing Tg. However, it is unusual in that addition of small amounts of water does not depress Tg, as in other sugars. Instead, a dihydrate crystal of trehalose forms, thereby shielding the remaining glassy trehalose from effects of the added water. Thus under less than ideal conditions such as high humidity and temperature, trehalose does indeed have special properties, which may explain the stability and longevity of anhydrobiotes that contain it. Further, it makes this sugar useful in stabilization of biomolecules of use in human welfare.

Expression of high-affinity trehalose-H+ symport in Saccharomyces cerevisiae.

The expression of the high-affinity trehalose-H+ symport was investigated in various Saccharomyces cerevisiae strains and culture conditions. Previous kinetic studies of trehalose transport in yeast have revealed the existence of at least two different uptake mechanisms: a high-affinity trehalose-H+ symport activity repressed by glucose, and a constitutive low-affinity transport activity, a putative facilitated diffusion process. Exogenously added trehalose was not an inducer of the high-affinity transport activity, and a correlation between trehalose and maltose uptake by yeast cells was found. Our results indicate that the maltose-H+ symporters encoded by MAL11, MAL21, and MAL41 are not responsible for the trehalose transport activity. The analysis of both trehalose and maltose transport activities in wild-type and in laboratory strains with defined MAL genes showed that the trehalose-H+ symporter was under control of MAL regulatory genes. Our results also suggest that the recently characterized AGT1 gene of S. cerevisiae may encode the high-affinity trehalose-H+ symporter. During diauxic growth on glucose the transport activity was low during the first exponential phase of growth, increased as glucose was exhausted from the medium, and decreased again as the cells reached the late stationary phase. This pattern was coincident with that of the intracellular levels of trehalose. The strong correlation between these two parameters may be of physiological significance during adaptation of yeast cells to stress conditions.

Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose: the role of vitrification.

R. P. Goodrich and co-workers (1989, U.S. Patent 4,874,690; 1992, Proc. Natl. Acad. Sci. USA 89,967-971) have reported that red blood cells can be preserved in the dry state by addition of mixtures of hydroxyethyl starch (HES) and glucose. More recently, Spieles and co-workers (1996, Cryo-Lett. 17, 43-52) found that HES alone is insufficient to preserve the dry cells and concluded on this basis that the studies of Goodrich et al. were incorrect. In the present paper we revisit that suggestion, using liposomes as a model to study effects of HES and glucose on membrane stability. In previous studies we and others have established that liposomes can be stabilized in the dry state if they are dried in the presence of disaccharides. Monosaccharides have not been effective. Measurements of effects of glucose on phase transitions in the dry lipids and vibrational frequency of the phosphate headgroup suggest that glucose shows an interaction with dry egg phosphatidylcholine similar to that seen with disaccharides. Nevertheless, glucose does not inhibit fusion in liposomes during drying, and it does not prevent leakage. Hydroxyethyl starch, which has a very high glass transition (Tg), inhibits fusion in the dry liposomes, but it does not depress the liquid crystalline to gel phase transition temperature (Tm) in the dry phospholipids, does not cause a shift in the phosphate vibration indicative of hydrogen bonding of the sugar to the phosphate, and does not stop leakage of trapped carboxyfluorescein. However, if glucose is added to the HES-containing samples, the liposomes are stabilized, so long as the samples are maintained below the Tg of the mixture. If they are heated above that Tg they fuse and leak their contents. We conclude that both glass formation and depression of Tm in the dry lipids are required. The role of glass formation in stabilization during drying of liposomes appears to be inhibition of fusion.

Is trehalose special for preserving dry biomaterials?

Simple sugars, especially disaccharides, stabilize biomaterials of various composition during air-drying or freeze-drying. We and others have provided evidence that direct interaction, an interaction that we believe is essential for the stabilization, between the sugar and polar groups in, for example, proteins and phospholipids occurs in the dry state. Some researchers, however, have suggested that the ability of the sugar to form a glass is the only requirement for stabilization. More recently, we have shown that both glass formation and direct interaction of the sugar and headgroup are often required for stabilization. In the present study, we present a state diagram for trehalose glass and suggest that the efficacy of this sugar for stabilization may be related to its higher glass transition temperatures at all water contents. We also show that trehalose and trehalose:liposome preparations form trehalose dihydrate as well as trehalose glass when rehydrated with water vapor. Formation of the dihydrate sequesters water, which might otherwise participate in lowering the glass transition temperature to below ambient. Because samples remain in the glassy state at ambient temperatures, viscosity is high and fusion between liposomes is prevented.

Membrane phase transition of intact human platelets: correlation with cold-induced activation.

Using Fourier transform infrared spectroscopy (FTIR), we have determined the phase transition temperature (Tm) of lipids in intact human platelets and have shown that it occurs between 15 and 18 degrees C, the temperature at which cold activation of platelets has previously been reported (Zucker and Borrelli, 1954, Blood, 28:602-608; White and Krivit, 1967, Blood, 30:625-635). The temperature at which the platelets pass through Tm is highly correlated with initial platelet shape change. However, shape change continues after the cells have passed through the phase transition. Cold-induced activation has previously prevented long-term storage of platelets at 4 degrees C. Antifreeze glycoproteins (AFGPs) isolated from polar fishes previously have been used to prevent ice crystal growth during freezing of tissues as well as leakage of solutes from liposomes as they were chilled through their Tm. We sought to determine if these AFGPs were able to stabilize platelets for long-term storage at 4 degrees C. Incubating platelets with antifreeze glycoproteins during long-term storage and rapid rewarming to 37 degrees C abrogated granule secretion associated with cold activation in a dose-dependent manner. This work suggests that AFGPs may be a possible solute for use in long-term low temperature storage of platelets.

Arbutin inhibits PLA2 in partially hydrated model systems.

Arbutin is a glycosylated hydroquinone found at high concentrations in certain plants capable of surviving extreme and sustained dehydration. In this paper, we examine a potential role of this molecule in anhydrobiosis. We have studied its effects on the physical properties of phospholipids and on preservation of liposomes during drying. Arbutin depresses the gel to liquid crystalline phase transition temperature of dry phospholipids, as measured by differential scanning calorimetry, with a pattern similar to that seen in phospholipids dried with the disaccharide trehalose. Unlike trehalose, however, arbutin does not protect dry liposomes from leaking their contents. Also, using Fourier transform infrared spectroscopy, we found an increase in the vibrational frequency of the phosphate asymmetric stretch in partially hydrated phospholipids in the presence of arbutin. Trehalose, by contrast, depresses the frequency of the phosphate in dry phospholipids, indicating that the modes of interaction of trehalose and arbutin with the bilayer are different. Previously, we have shown that phospholipases can be active in liposomes with surprisingly low water contents. Based on the structural similarity of arbutin to a known inhibitor of phospholipase A2 (PLA2), it appeared possible that arbutin might serve as an inhibitor of phospholipases. Liposomes of varying composition were lyophilized in the presence and absence of phospholipases. When the liposomes were partially rehydrated at 76% relative humidity, arbutin inhibited PLA2, but did not inhibit phospholipases B or C. Accumulation of enzyme product in the liposome membranes was measured by analytical thin layer chromatography, and was taken as a measure of enzyme activity. Arbutin did not inhibit any of the enzymes in the presence of excess water. Based on these data, hypotheses are presented concerning the mechanism of PLA2 inhibition by arbutin in the mostly dehydrated state.

Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions.

Antifreeze glycoproteins (AFGPs), found in the blood of polar fish at concentrations as high as 35 g/liter, are known to prevent ice crystal growth and depress the freezing temperature of the blood. Previously, Rubinsky et al. [Rubinsky, B., Mattioli, M., Arav, A., Barboni, B. & Fletcher, G. L. (1992) Am. J. Physiol. 262, R542-R545] provided evidence that AFGPs block ion fluxes across membranes during cooling, an effect that they ascribed to interactions with ion channels. We investigated the effects of AFGPs on the leakage of a trapped marker from liposomes during chilling. As these liposomes are cooled through the transition temperature, they leak approximately 50% of their contents. Addition of less than 1 mg/ml of AFGP prevents up to 100% of this leakage, both during chilling and warming through the phase transition. This is a general effect that we show here applies to liposomes composed of phospholipids with transition temperatures ranging from 12 degrees C to 41 degrees C. Because these results were obtained with liposomes composed of phospholipids alone, we conclude that the stabilizing effects of AFGPs on intact cells during chilling reported by Rubinsky et al. may be due to a nonspecific effect on the lipid components of native membranes. There are other proteins that prevent leakage, but only under specialized conditions. For instance, antifreeze proteins, bovine serum albumin, and ovomucoid all either have no effect or actually induce leakage. Following precipitation with acetone, all three proteins inhibited leakage, although not to the extent seen with AFGPs. Alternatively, there are proteins such as ovotransferrin that have no effect on leakage, either before or after acetone precipitation.