Two cytochrome P450 (CYP), CYP1A1 and CYP1A2, cDNA sequences have been isolated and cloned from harp seal (Phoca groenlandica) and grey seal (Halichoerus grypus). EROD, a model substrate for CYP1A, and heterologous antibodies have been employed as a biomarker in marine mammals, however the CYP1A sequences have not been characterised in these two seal species. mRNA was used as the template in RT-PCR, rather than DNA as this indicates transcription of the CYP1A gene in these seal species exposed to environmental contaminants. Harp and grey seal CYP1A1 amino acid sequences exhibited >99% identity and the CYP1A2 sequences were >98% identical. Phylogenetic analyses of the two seal species with other mammalian, and avian CYP1A sequences, showed the CYP1A1 and CYP1A2 sequences clustered with corresponding sequences in other mammalian species. The closest sequences to the seal CYP1As was dog CYP1A. The CYP1A sequence information presented in this study has provided the necessary data for the future production of species-specific probes for the use as biomarkers of environmental contaminant exposure.

We demonstrate a novel concept for a position sensitive microchannel plate detector. This detector provides sub-millimeter spatial resolution by examining the signal induced on a wire harp by the electron cloud from a microchannel plate detector. Wires in the harp are efficiently read out by coupling them to a delay line.

OSF-1 (osteoblast-specific factor-1), which is also referred to as p18, HBBM, HB-GAM, HBGF-8, HARP, HBNF, and pleiotrophin, is a 121-amino acid polypeptide that can induce neurite outgrowth in vitro and is highly expressed in several tissues during fetal development but exhibits expression restricted to brain and bone tissues in adults. We have reported the genomic structure of mouse OSF-1 gene, in which the open reading frame spans four exons and at least two additional 5'-UTR exons (upstream exon U2 and downstream exon U1) exist. From analysis of isolated cDNAs, two types of cDNAs were identified: one has a sequence for U1 and U2 and the other has a sequence for an intron (present between U1 and U2) and U1. This suggests that the OSF-1 gene utilizes two alternative promoters, a distal and a proximal promoter, designated promoters II and I, respectively, for the translation initiation site (ATG). Promoter II is thought to exist upstream of the intron, while promoter I is present in the intron. RT-PCR was employed to examine which OSF-1 promoters are used during development and in various cell lines. In adult mice (aged 2 months), usage of promoter I was predominant, and OSF-1 mRNAs were expressed in many organs including brain and bone. At one fetal stage (E-19), promoter I was active in the major organs including brain, liver, kidney, and intestine, while promoter II was active only in the brain. In the cell lines examined, usage of promoter I was frequent, while promoter II was active only in a few cell lines such as MC3T3-E1 (cultured for 7 days) and C3H10T1/2. These findings suggest that OSF-1 may play fundamental roles in differentiation, growth and maintenance of adult organs as well as in embryogenesis, and indicate that the expression of OSF-1 is regulated, at least in part, by the usage of different promoters in the mouse.

We provide and evaluate an open-source software solution for automatically measuring hippocampal volume and hippocampal surface roughness based on T1-weighted MRI, which allows for discriminating between patients with Alzheimer's disease (AD) or mild cognitive impairment (MCI) and elderly controls (NC) using only one scan.

This solution is based on a fast multiple-atlas segmentation technique, which combines a patch-based labeling method with an atlas-warping using non-rigid registrations.

The classifications are comparable to the best classifications in a large clinical dataset. For AD vs control, we obtain a high degree of accuracy, approximately 90%. For MCI vs control, we obtain accuracies ranging from 70% to 78%. The average time for the hippocampal segmentation from a T1-MRI is less than 17min.

In this study, we investigate a combination of our method with annotations using the Harmonized Hippocampal Protocol (HarP). We compare its capabilities with the FreeSurfer method and verify its impact on segmentation and diagnostic group separation capabilities. Our approach is developed and validated using 134 subjects from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database with annotations from HarP. Then, this method, tuned with the best parameters, is applied to 162 subjects from a private image database.

Our approach with HarP annotations has a high level of accuracy for segmentation of the hippocampus and is robust to multi-site data. The bio-markers extracted from our proposed method have discriminative power based on a scalar feature, showing robustness in generalization and avoid overfitting. The computational time in our hippocampal segmentation algorithm has decreased considerably compared to other available analysis.

This study examined the concomitant use of 2 complementary and alternative medicine modalities, Healing Harp and Healing Touch, to reduce pain, anxiety, and nausea in the postoperative patient population. The results demonstrate the effectiveness of using concomitant Healing Touch and Healing Harp to significantly reduce moderate to severe pain and anxiety in this patient population. Further research is warranted.

Cardiac magnetic resonance imaging (MRI) provides information about myocardial morphology, function, and viability from cine, tagged, and late gadolinium enhancement (LGE) images, respectively. While the cine and tagged images are acquired in a time-resolved fashion, the LGE images are acquired at a single timeframe. The purpose of this work is to develop a method for generating cine LGE images without additional scan time. The motion field is extracted from the tagged images, and is then used to guide the deformation of the infarcted region from the acquired LGE image at the acquired timeframe to any other timeframe. Major techniques for motion estimation, including harmonic phase (HARP) and optical flow analysis, are tested in this work for motion estimation. The proposed method is tested on numerical phantom and images from four human subjects. The generated cine LGE images showed both viability and wall motion information in the same set of images without additional scan time or image misregistration problems. The band-pass optical flow analysis resulted in the most accurate motion estimation compared to other methods, especially HARP, which fails to track points at the myocardial boundary. Infarct transmurality from the generated images showed good agreement with myocardial strain, and wall thickening showed good agreement with that measured from conventional cine images. In conclusion, the developed technique allows for generating cine LGE images that enable simultaneous display of wall motion and viability information. The generated images could be useful for estimating myocardial contractility reserve and for treatment prognosis.

Recently, a group of major international experts have completed a comprehensive effort to efficiently define a harmonized protocol for manual hippocampal segmentation that is optimized for Alzheimer's research (known as the EADC-ADNI Harmonized Protocol (the HarP)). This study compares the HarP with one of the widely used hippocampal segmentation protocols (Pruessner, 2000), based on a single automatic segmentation method trained separately with libraries made from each manual segmentation protocol. The automatic segmentation conformity with the corresponding manual segmentation and the ability to capture Alzheimer's disease related hippocampal atrophy on large datasets are measured to compare the manual protocols. In addition to the possibility of harmonizing different procedures of hippocampal segmentation, our results show that using the HarP, the automatic segmentation conformity with manual segmentation is also preserved (Dice's κ=0.88,κ=0.87 for Pruessner and HarP respectively (p = 0.726 for common training library)). Furthermore, the results show that the HarP can capture the Alzheimer's disease related hippocampal volume differences in large datasets. The HarP-derived segmentation shows large effect size (Cohen's d = 1.5883) in separating Alzheimer's Disease patients versus normal controls (AD:NC) and medium effect size (Cohen's d = 0.5747) in separating stable versus progressive Mild Cognitively Impaired patients (sMCI:pMCI). Furthermore, the area under the ROC curve for a LDA classifier trained based on age, sex and HarP-derived hippocampal volume is 0.8858 for AD:NC, and for 0.6677 sMCI:pMCI. These results show that the harmonized protocol-derived labels can be widely used in clinic and research, as a sensitive and accurate way of delineating the hippocampus.

Regulatory T cells (Tregs) are key players in the regulation of inflammatory responses. In this study, two natural molecules, namely, sparteine sulfate (SS) and harpagoside (Harp), were investigated for their ability to induce Tregs in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy volunteers and grown in the presence or absence of ConA, with TGF-beta, SS or Harp. Expression of the mRNA of FoxP3, TGF-beta, IL-10 and GAPDH was assessed via q-PCR. The expression of Treg markers including CD4, CD25, CD127 and FoxP3 was measured via flow cytometry. The secretion of IL-10 and TGF-beta by cultured cells was assessed by ELISA. Furthermore, the suppressive role of SS and Harp on PBMCs in vitro was tested via allogeneic mixed lymphocyte reaction (MLR). Data obtained show that both compounds increased the expression of FoxP3, TGF-beta and IL-10 mRNA in resting PBMCs but to a lesser extent in activated cells. Moreover, they significantly increased the percent of CD4⁺CD25⁺FoxP3⁺CD127^- Tregs in activated and naïve PBMCs. Functionally, both compounds caused a significant reduction in the stimulation index in allogeneic MLR. Together, our data demonstrate for the first time that SS and Harp can induce human Tregs in vitro and therefore have great potential as anti-inflammatory agents.

Keywords: CD4+CD25+ regulatory T cells; Foxp3 induction; human; mixed lymphocyte reaction (MLR); natural molecules.

Our understanding of vibrissal function in pinnipeds is poor due to the lack of comparative morphological, neurobiological, and psychophysical performance data. In contrast, the function of terrestrial mammalian vibrissae is better studied. Pinnipeds have the largest vibrissae of all mammals, and phocids may have the most modified vibrissae. The tactile performance for pinniped vibrissae is well known for harbor seals (Phoca vitulina). Harbor seals display at least two types of tactile behavior involving their mystacial vibrissae: a fine discriminatory capability using active touch and hydrodynamic trail following (the ability to detect and follow turbulent trails). This study investigated innervation patterns of harbor seal follicle-sinus complexes (F-SCs) to test the hypothesis that the whiskers used in hydrodynamic trail following possess increased innervation investment compared to other phocids. Therefore, the most lateral vibrissae from five harbor seals were histologically processed so that morphometric measurements and axon counts could be collected. Vibrissae from one harbor seal were immunolabeled with anti-protein gene product (PGP 9.5) to document the pattern of deep vibrissal nerve innervation of the F-SCs. Overall, harbor seals showed an innervation pattern (axons/F-SC and axons/muzzle) similar to other phocids. The ventrolateral vibrissae, involved in hydrodynamic trail following, have greater axon density in harbor seals than harp seals, suggesting harbor seal F-SC innervation patterns could explain their performance at trail following. The combination of microstructural, innervation investment, and behavioral data provides a foundation for functional inference regarding this tactile behavior in harbor seals and also facilitates future comparative work for other pinniped species. Anat Rec, 302:1837-1845, 2019. © 2019 American Association for Anatomy.

Assessment of the compressibility of marine mammal airways at depth is crucial to understanding vital physiologic processes such as gas exchange during diving. Very few studies have directly assessed changes in cetacean and pinniped trachea-bronchial shape, and none have quantified changes in volume with increasing pressure. A freshly deceased harbor seal, grey seal, harp seal, harbor porpoise, and common dolphin were imaged post mortem via CT in a radiolucent hyperbaric chamber as previously described in Moore et al (2011). Volume reconstructions were performed of segments of the trachea and bronchi of the pinnipeds and bronchi of the cetaceans for each pressure treatment. All specimens examined demonstrated significant decreases in volume with increasing pressure, with the harbor seal and common dolphin nearing complete collapse at the highest pressures. The common dolphin bronchi demonstrated distinctly different compression dynamics between 50% and 100% lung inflation treatments, indicating the importance of air in maintaining patent airways, and collapse occurred caudally to cranially in the 50% treatment. Dynamics of the harbor seal and grey seal airways indicated that the trachea was less compliant than the bronchi. These findings indicate potential species-specific variability in airway compliance, and cessation of gas exchange may occur at greater depths than those predicted in models assuming rigid airways. This may potentially increase the likelihood of decompression sickness in these animals during diving.

The mature 5'-ends of tRNAs are generated by RNase P in all domains of life. The ancient form of the enzyme is a ribonucleoprotein consisting of a catalytic RNA and one or more protein subunits. However, in the hyperthermophilic bacterium Aquifex aeolicus and close relatives, RNase P is a protein-only enzyme consisting of a single type of polypeptide (Aq_880, ~23 kDa). In many archaea, homologs of Aq_880 were identified (termed HARPs for Homologs of Aquifex RNase P) in addition to the RNA-based RNase P, raising the question about the functions of HARP and the classical RNase P in these archaea. Here we investigated HARPs from two euryarchaeotes, Haloferax volcanii and Methanosarcina mazei. Archaeal strains with HARP gene knockouts showed no growth phenotypes under standard conditions, temperature and salt stress (H. volcanii) or nitrogen deficiency (M. mazei). Recombinant H. volcanii and M. mazei HARPs were basically able to catalyse specific tRNA 5'-end maturation in vitro. Furthermore, M. mazei HARP was able to rescue growth of an Escherichia coli RNase P depletion strain with comparable efficiency as Aq_880, while H. volcanii HARP was unable to do so. In conclusion, both archaeal HARPs showed the capacity (in at least one functional assay) to act as RNases P. However, the ease to obtain knockouts of the singular HARP genes and the lack of growth phenotypes upon HARP gene deletion contrasts with the findings that the canonical RNase P RNA gene cannot be deleted in H. volcanii, and a knockdown of RNase P RNA in H. volcanii results in severe tRNA processing defects. We conclude that archaeal HARPs do not make a major contribution to global tRNA 5'-end maturation in archaea, but may well exert a specialised, yet unknown function in (t)RNA metabolism. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1109-1116, 2019.

Cardiac magnetic resonance (MR) tissue tagging offers an excellent solution for tracking deformation and is considered the reference standard for the quantification of strain. However, due to the requirements for a dedicated acquisition sequence and post-processing software, tagged MR acquisitions are performed much less frequently in routine clinical practice than the anatomical cine MR sequence. Using tagged MR as the reference standard, this study proposes an approach to evaluate a diffeomorphic image registration algorithm applied on cine MR images to compute the cardiac deformation. In contrast to previous evaluation methods that compared the final results, such as strain, computed from cine and tagged MR sequences, the proposed method performs a direct frame-to-frame comparison in the evaluation. To overcome the problem of misalignment between the tagged and cine MR images, the proposed approach performs transformations to and from the two-dimensional image pixel coordinates and three-dimensional space using the meta-information encoded in the MR images. Linear temporal interpolation is performed using the frame acquisition time since the last R-wave peak value of the electrocardiogram signal recorded in the meta-information. Several statistic measures are computed and reported for the registration error using the Euclidean distances between the corresponding set of points obtained using cine and tagged MR images.

In arid yet foggy regions, fog harvesting is emerging as a promising approach to combat water scarcity. The mesh netting used by current fog harvesters suffers from inefficient drainage, which severely constrains the water collection efficiency. Recently, it was demonstrated that fog harps can significantly enhance water harvesting as the vertical wire array does not obstruct the drainage pathway. However, fabrication limitations resulted in a very low shade coefficient of 18% for the initial fog harp prototype and the field testing was geographically confined to light fog conditions. Here, we use wire-electrical discharge machining (wire-EDM) to machine ultrafine comb arrays; winding the harp wire along a comb-embedded reinforced frame enabled a shade coefficient of 50%. To field test under heavy fog conditions, we placed the harvesters on a closed-circuit test road and inundated them with fog produced by an array of overlying fog towers. On average, the fog harps collected about three times more water than the mesh netting. During fog harvesting, the harp wires were observed to tangle together due to the surface tension of water. We developed a rational model to predict the extent of the tangling problem for any given fog harp design. By designing next-generation fog harps to be anti-tangling, we expect that even larger performance multipliers will be possible compared to the current mesh harvesters.

Keywords: anti-clogging; droplet; elastocapillarity; field test; fog harp; fog harvesting; wire tangling.

Results of investigation of mineral levels in the harp seal pups blood serum in connection with adaptation to the captivity are presented. The contents of copper, zinc, iron, magnesium, cobalt, manganese, sodium, potassium, calcium and phosphorus in the first 18 day of animals keeping in the oceanarium were determined. The causes of the revealed differences in changes of the contents of single elements during the observation term, and also question on duration of fading stress alterations and stabilization of biochemical parameters of marine mammals blood are discussed.

Three types of fibre, dark (type 1), light (type 2) and intermediate, were distinguished in the caval sphincter muscle of the diaphragm in the harp seal (Pagophilus groenlandicus) using histochemical and electron-microscopic techniques. The dark fibre contained large peripheral aggregations of mitochondria, numerous lipid droplets and dense aggregates of glycogen granules. The same features were observed in the dark fibre of the diaphragm muscle too. In the light of the low oxidative enzyme activity and high lipase activity observed in the diaphragm and other skeletal muscles of the harp seal in previous studies, it is postulated that these mitochondrial aggregations and lipid droplets represent an adaptation for the generation of heat through non-shivering thermogenesis comparable to that in the brown adipose tissue. Such ability to generate and maintain heat by uncoupling the oxidative phosphorylation process should enable the caval sphincter muscle to function efficiently in regulating the cardiac return of blood from the inferior vena cava during a dive when the body temperature as a whole drops. The lack of the regional differences in the fibre composition of the harp seal diaphragm, as were reported in the rat diaphragm, is attributed to the seal's large body size, lower metabolic rate and diving habit.

Hydrogen bonds (HB) in proteins and nucleic acids oscillate with frequencies of 10(11)-10(12) Hz, so giving rise to mm waves and far infrared emission. As HB undergo stretching and compression, their oscillation frequency will correspondingly change. The author (1989) 1b has devised a "musical" model, in which HB are compared to the strings of a string instrument: the harp. On the length, number and cooperation of "strings" (small and large hydrogen-harps) will depend the frequency, intensity and quality of "sound" (i.e. electromagnetic emission). Of course, this elementary hydrogen-harps model needs a physico-mathematical development. Nevertheless it can already be utilized to explain some essential moments of proteins and DNA function, on the base of its logical connection with Fröhlich's theory of coherent excitations. It might also help the understanding of the second genetic code mechanisms.

Commercial harvesting of snow crab (Chionoecetes opilio) in the Barents Sea started in 2012 by Norwegian fishing vessels. This new fishery has significant bait requirements, representing an emerging conservation challenge. In this study, we evaluate the performance of five alternative (natural) baits manufactured from the waste stream of existing and sustainably managed harp seal (Pagophilus groenlandicus) and minke whale (Balaenoptera acutorostrata) capture. Five different types of new bait were evaluated, including seal fat (SF), seal fat with skin (SFS), seal meat with bone (SMB), whale fat with skin (WFS), and whale meat with fat (WMF). A comparative fishing experiment was conducted onboard a commercial snow crab fishing vessel in the Barents Sea (May-June, 2016) to evaluate the performance of traditional bait (squid, Illexs spp.) and alternative baits at catching snow crabs. Performance of the different baits were compared on the basis of the number of commercial crab caught per trap haul catch per unit effort (CPUE) and carapace width (CW). Our results showed that SF and SFS performed equally well to traditional bait, with no statistical difference in CPUE (p-value = 0.325 and 0.069, respectively). All of the other experimental baits significantly decreased CPUE, when compared to squid. No significant effect of bait treatment on CW was detected and the cumulative distribution of CW was the same between control traps and each of the bait treatments. Overall the results indicated that SF and SFS represent a viable alternative to replace traditional bait, addressing a key conservation challenge in this bait intensive snow crab fishery.

The present study investigated how concentrations of different organochlorines (OCs) vary with nutritional condition in adult harp seal (Phoca groenlandica) females in a captive fasting experiment and in the wild. During the first part of this study seals in good condition (N= 5) were fasted for a period of 28 days and blood and blubber samples were collected at days 1, 14 and 28. Blubber OC concentrations remained unchanged throughout the experimental period, and were consistently significantly higher than concentrations in the blood. In contrast to blubber OC levels, blood OC levels showed a significant time-dependant increase. During the fasting experiment the seals lost an average of 24 kg of body mass, which is less than half the natural annual variation in this species in the wild. The second part of this study compared OC concentrations in blood and blubber from seals collected at prime condition before the breeding season (N = 10) with animals collected during molt when condition is poor (N = 7). The average mass difference between the two groups was more than 40 kg. Blood levels of most OCs were significantly higher in the thin seals compared with the levels found in the fat seals. These differences in blood OC concentrations were much greater than what was found during the fasting experiment. For example sigmaPCB levels in the blood during the fasting experiment increased by approximately 83%, while the corresponding increase in blood levels of sigmaPCB between the two other samples was 720% (from 201 to 1,447 ng/g lipid). Blubber levels of OCs from the animals in the wild were significantly higher in the thin seals compared with the fat animals. In addition, the blubber levels of OCs were significantly higher than blood levels of OCs only for the fat seals. This study demonstrates the extreme variability present in the concentrations of OCs in blood of seals in response to change in condition. Since the natural variation in condition is extreme during phocid seals' annual cycles, we recommend that blood should not be used in studies of OCs where the aim of the study is to monitor OC levels for comparative purposes or time-trend analysis.

We report the detection of a transiting planet around π Men (HD 39091), using data from the Transiting Exoplanet Survey Satellite (TESS). The solar-type host star is unusually bright (V = 5.7) and was already known to host a Jovian planet on a highly eccentric, 5.7-year orbit. The newly discovered planet has a size of 2.04 ± 0.05 R_⊕ and an orbital period of 6.27 days. Radial-velocity data from the HARPS and AAT/UCLES archives also displays a 6.27-day periodicity, confirming the existence of the planet and leading to a mass determination of 4.82±0.85 M_⊕. The star's proximity and brightness will facilitate further investigations, such as atmospheric spectroscopy, asteroseismology, the Rossiter-McLaughlin effect, astrometry, and direct imaging.

Because Korean society is fast becoming multi-ethnic, the determination of ambiguous human leukocyte antigen (HLA) types using HLA allele frequencies is becoming less applicable. In this study, we focused on the development of new technical methods to directly resolve the ambiguities arising from HLA genotyping. One hundred and fifty unrelated healthy Korean adults were included in this study. All alleles from each HLA locus were first divided into 2-4 groups, with each group amplified in a single PCR tube (multi-group-specific amplification, MGSA). To resolve phase ambiguities, some allele groups were also amplified separately in small group-specific amplification (SGSA) tubes. In order to then resolve incomplete sequence ambiguities, primers for MGSA and SGSA were initially designed to cover additional exons. If needed, a heterozygous ambiguity resolving primer (HARP) or sequence specific primer (SSP) was also used. When MGSA and SGSA methods were applied, the rate of phase ambiguity was greatly reduced to an average of 6% (1.3% in HLA-A, 15.7% in -B, and 2.0% in -C). Additional HARP and SSP methods could resolve all the phase ambiguities. Using our proposed method, we also detected three alleles that have not been previously reported in Korea, C*04:82, C*07:18, and C*08:22, and report 6-digit level HLA allele and haplotype frequencies among Koreans. In conclusion, the use of MGSA/SGSA for the initial amplification step is a cost-effective method facilitating timely and accurate reporting, given the continuing increase in the ethnic diversity of the Korean population. The MGSA described here can be applicable to various populations and thus could be shared by the majority of HLA typing laboratories. However, efforts to solve HLA ambiguity should continue, because SGSA, HARPs and SSPs would be specific to a particular population.

In the Barents Sea, pelagic and coastal polar bears are facing various ecological challenges that may explain the difference in their pollutant levels. We measured polychlorinated biphenyls, organochlorine pesticides, polybrominated diphenyl ethers in fat, and perfluoroalkyl substances in plasma in pelagic and coastal adult female polar bears with similar body condition. We studied polar bear feeding habits with bulk stable isotope ratios of carbon and nitrogen. Nitrogen isotopes of amino acids were used to investigate their trophic position. We studied energy expenditure by estimating field metabolic rate using telemetry data. Annual home range size was determined, and spatial gradients in pollutants were explored using latitude and longitude centroid positions of polar bears. Pollutant levels were measured in harp seals from the Greenland Sea and White Sea-Barents Sea as a proxy for a West-East gradient of pollutants in polar bear prey. We showed that pelagic bears had higher pollutant loads than coastal bears because (1) they feed on a higher proportion of marine and higher trophic level prey, (2) they have higher energy requirements and higher prey consumption, (3) they forage in the marginal ice zones, and (4) they feed on prey located closer to pollutant emission sources/transport pathways.

Liver and/or muscle samples were collected from harp seals on the breeding and moulting grounds in the White Sea area (N = 316) and in the Greenland Sea (N = 225). Of a total of 25 presumptive loci detected by isoelectric focusing on polyacrylamide gels (IFPAG), 11 were variable. Of these, 4 were polymorphic at the 95% level, another 2 at the 99% level. The study shows that the higher resolving capacity obtained by IFPAG, as compared to starch gel electrophoresis, increases the number of electrophoretically detectable polymorphic loci in this species. The paper also compiles a set of methodical modifications which adapt IFPAG to large scale multilocus screening. The study indicates no marked differences between the White Sea and Greenland Sea stocks in frequency distributions of the consistently resolved enzyme systems. Other evidence suggests that potential exchange of genetic material between Northeast Atlantic harp seal stocks may still be so limited that for management purposes the stocks may be regarded as separate and self-sustaining units.