The effects of facioscapulohumeral dystrophy and dynamic arm support on upper extremity muscle coordination in functional tasks.

This study's objective is to understand the effect of muscular weakness in persons with facioscapulohumeral dystrophy as well as the effect of a dynamic arm support on muscle coordination and activity performance, during activities of daily living. People with facioscapulohumeral dystrophy (n=12, 56.0±14.5 years) and healthy controls (n=12, 55.5±13.4 years) performed five simulated daily activity tasks, while unsupported and supported by the Gowing dynamic arm support. Surface electromyography, kinematics, and maximum force output were recorded. Outcomes were calculated for muscle coordination (muscle synergies), maximum muscle activity, movement performance indicators, and upper limb muscular weakness (maximum force output). Muscle coordination was altered and less consistent in persons with facioscapulohumeral dystrophy compared with healthy controls. The dynamic arm support alleviated muscle efforts and affected muscle coordination in both populations. While populations became more similar, the internal consistency of persons with facioscapulohumeral dystrophy remained unaffected and lower than that of healthy controls. Furthermore, the support affected movements' performance in both groups. The maximum force outputs were lower in persons with facioscapulohumeral dystrophy than controls. Muscle coordination differences were presumably the result of individual-specific in muscle weakness and compensatory strategies for dealing with gravity compensation and movement constraints.

Lower limb muscle fatigue after uphill walking in children with unilateral spastic cerebral palsy.

Fatigue during walking is a common complaint in cerebral palsy (CP). The primary purpose of this study is to investigate muscle fatigue from surface electromyography (sEMG) measurements after a treadmill-based fatigue protocol with increasing incline and speed in children with CP with drop foot. The secondary purpose is to investigate whether changes in sagittal kinematics of hip, knee and ankle occur after fatigue. Eighteen subjects with unilateral spastic CP performed the protocol while wearing their ankle-foot orthosis and scored their fatigue on the OMNI scale of perceived exertion. The median frequency (MF) and root mean square (RMS) were used as sEMG measures for fatigue and linear mixed effects model were applied. The MF was significantly decreased in fatigued condition, especially in the affected leg and in the tibialis anterior and peroneus longus muscle. The RMS did not change significantly in fatigued condition, while the OMNI fatigue score indicated patients felt really fatigued. No changes in sagittal kinematics of hip, knee and ankle were found using statistical non-parametric mapping. In conclusion, the current fatigue protocol seems promising in inducing fatigue in a population with CP with drop foot and it could be used to expand knowledge on muscle fatigue during walking in CP.

Recommendations for studies on dynamic arm support devices in people with neuromuscular disorders: a scoping review with expert-based discussion.

PURPOSE: Neuromuscular disorders are characterised by muscle weakness that limits upper extremity mobility, but can be alleviated with dynamic arm support devices. Current research highlights the importance and difficulties of evidence-based recommendations for device development. We aim to provide research recommendations primarily concerning upper extremity body functions, and secondarily activity and participation, environmental and personal factors. METHODS: Evidence was synthesised from literature, ongoing studies, and expert opinions and tabulated within a framework based on a combination of the International Classification of Functioning, Disability and Health (ICF) model and contextual constructs. RESULTS: Current literature mostly investigated the motor capacity of muscle function, joint mobility, and upper body functionality, and a few studies also addressed the impact on activity and participation. In addition, experts considered knowledge on device utilisation in the daily environment and characterising the beneficiaries better as important. Knowledge gaps showed that ICF model components and contextual constructs should be better integrated and more actively included in future research. CONCLUSIONS: It is recommended to, first, integrate multiple ICF model components and contextual constructs within one study design. Second, include the influence of environmental and personal factors when developing and deploying a device. Third, include short-term and long-term measurements to monitor adaptations over time. Finally, include user satisfaction as guidance to evaluate the device effectiveness.IMPLICATIONS ON REHABILITATIONSynthesized evidence will support future research and development of dynamic arm supports.Tabulated evidence stresses the importance of integrating ICF model components and contextual constructs to fill the knowledge gaps.Presented knowledge gaps and proposed steps guide the set up of future studies on dynamic arm supports.

Inflammation and TGF-ß Signaling Differ between Abdominal Aneurysms and Occlusive Disease.

Abdominal aortic aneurysms (AAA), are usually asymptomatic until rupture causes fatal bleeding, posing a major vascular health problem. AAAs are associated with advanced age, male gender, and cardiovascular risk factors (e.g. hypertension and smoking). Strikingly, AAA and AOD (arterial occlusive disease) patients have a similar atherosclerotic burden, yet develop either arterial dilatation or occlusion, respectively. The molecular mechanisms underlying this diversion are yet unknown. As this knowledge could improve AAA treatment strategies, we aimed to identify genes and signaling pathways involved. We compared RNA expression profiles of abdominal aortic AAA and AOD patient samples. Based on differential gene expression profiles, we selected a gene set that could serve as blood biomarker or as pharmacological intervention target for AAA. In this AAA gene list we identified previously AAA-associated genes COL11A1, ADIPOQ, and LPL, thus validating our approach as well as novel genes; CXCL13, SLC7A5, FDC-SP not previously linked to aneurysmal disease. Pathway analysis revealed overrepresentation of significantly altered immune-related pathways between AAA and AOD. Additionally, we found bone morphogenetic protein (BMP) signaling inhibition simultaneous with activation of transforming growth factor ß (TGF-ß) signaling associated with AAA. Concluding our gene expression profiling approach identifies novel genes and an interplay between BMP and TGF-ß signaling regulation specifically for AAA.

Defective Connective Tissue Remodeling in Smad3 Mice Leads to Accelerated Aneurysmal Growth Through Disturbed Downstream TGF-ß Signaling.

Aneurysm-osteoarthritis syndrome characterized by unpredictable aortic aneurysm formation, is caused by SMAD3 mutations. SMAD3 is part of the SMAD2/3/4 transcription factor, essential for TGF-ß-activated transcription. Although TGF-ß-related gene mutations result in aneurysms, the underlying mechanism is unknown. Here, we examined aneurysm formation and progression in Smad3-/- animals. Smad3-/- animals developed aortic aneurysms rapidly, resulting in premature death. Aortic wall immunohistochemistry showed no increase in extracellular matrix and collagen accumulation, nor loss of vascular smooth muscle cells (VSMCs) but instead revealed medial elastin disruption and adventitial inflammation. Remarkably, matrix metalloproteases (MMPs) were not activated in VSMCs, but rather specifically in inflammatory areas. Although Smad3-/- aortas showed increased nuclear pSmad2 and pErk, indicating TGF-ß receptor activation, downstream TGF-ß-activated target genes were not upregulated. Increased pSmad2 and pErk staining in pre-aneurysmal Smad3-/- aortas implied that aortic damage and TGF-ß receptor-activated signaling precede aortic inflammation. Finally, impaired downstream TGF-ß activated transcription resulted in increased Smad3-/- VSMC proliferation. Smad3 deficiency leads to imbalanced activation of downstream genes, no activation of MMPs in VSMCs, and immune responses resulting in rapid aortic wall dilatation and rupture. Our findings uncover new possibilities for treatment of SMAD3 patients; instead of targeting TGF-ß signaling, immune suppression may be more beneficial.

Fibulin-4 deficiency increases TGF-ß signalling in aortic smooth muscle cells due to elevated TGF-ß2 levels.

Fibulins are extracellular matrix proteins associated with elastic fibres. Homozygous Fibulin-4 mutations lead to life-threatening abnormalities such as aortic aneurysms. Aortic aneurysms in Fibulin-4 mutant mice were associated with upregulation of TGF-ß signalling. How Fibulin-4 deficiency leads to deregulation of the TGF-ß pathway is largely unknown. Isolated aortic smooth muscle cells (SMCs) from Fibulin-4 deficient mice showed reduced growth, which could be reversed by treatment with TGF-ß neutralizing antibodies. In Fibulin-4 deficient SMCs increased TGF-ß signalling was detected using a transcriptional reporter assay and by increased SMAD2 phosphorylation. Next, we investigated if the increased activity was due to increased levels of the three TGF-ß isoforms. These data revealed slightly increased TGF-ß1 and markedly increased TGF-ß2 levels. Significantly increased TGF-ß2 levels were also detectable in plasma from homozygous Fibulin-4(R/R) mice, not in wild type mice. TGF-ß2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation. In conclusion, we have shown increased TGF-ß signalling in isolated SMCs from Fibulin-4 deficient mouse aortas, not only caused by increased levels of TGF-ß1, but especially TGF-ß2. These data provide new insights in the molecular interaction between Fibulin-4 and TGF-ß pathway regulation in the pathogenesis of aortic aneurysms.

An inverse dynamic analysis on the influence of upper limb gravity compensation during reaching.

Muscular dystrophies (MDs) are characterized by progressive muscle wasting and weakness. Several studies have been conducted to investigate the influence of arm supports in an attempt to restore arm function. Lowering the load allows the user to employ the residual muscle force for movement as well as for posture stabilization. In this pilot study three conditions were investigated during a reaching task performed by three healthy subjects and three MD subjects: a control condition involving reaching; a similar movement with gravity compensation using braces to support the forearm; an identical reaching movement in simulated zero-gravity. In the control condition the highest values of shoulder moments were present, with a maximum of about 6 Nm for shoulder flexion and abduction. In the gravity compensation and zero gravity conditions the maximum shoulder moments were decreased by more than 70% and instead of increasing during reaching, they remained almost unvaried, fluctuating around an offset value less than 1 Nm. Similarly, the elbow moments in the control condition were the highest with a peak around 3.3 Nm for elbow flexion, while the moments were substantially reduced in the remaining two conditions, fluctuating around offset values between 0 to 0.5 Nm. In conclusion, gravity compensation by lower arm support is effective in healthy subjects and MD subjects and lowers the amount of shoulder and elbow moments by an amount comparable to a zero gravity environment. However the influence of gravity compensation still needs to be investigated on more people with MDs in order to quantify any beneficial effect on this population.

Rad54 is required for the normal development of male and female germ cells and contributes to the maintainance of their genome integrity after genotoxic stress.

Rad54 is an important factor in the homologous recombination pathway of DNA double-strand break repair. However, Rad54 knockout (KO) mice do not exhibit overt phenotypes at adulthood, even when exposed to radiation. In this study, we show that in Rad54 KO mouse the germline is actually altered. Compared with the wild-type (WT) animals, these mice have less premeiotic germ cells. This germ cell loss is found as early as in E11.5 embryos, suggesting an early failure during mutant primordial germ cells development. Both testicular and ovarian KO germ cells exhibited high radiation sensitivity leading to a long-term gametogenesis defect at adulthood. The KO female germline was particularly affected displaying decreased litter size or sterility. Spermatogenesis recovery after irradiation was slower and incomplete in Rad54 KO mice compared with that of WT mice, suggesting that loss of germ stem cell precursors is not fully compensated along the successive rounds of spermatogenesis. Finally, spermatogenesis recovery after postnatal irradiation is in part regulated by glial-cell-line-derived neurotrophic factor (GDNF) in KO but not in irradiated WT mice, suggesting that Sertoli cell GDNF production is stimulated upon substantial germ cell loss only. Our findings suggest that Rad54 has a key function in maintaining genomic integrity of the developing germ cells.

Opposite modifying effects of HR and NHEJ deficiency on cancer risk in Ptc1 heterozygous mouse cerebellum.

Heterozygous Patched1 (Ptc1(+/-)) mice are prone to medulloblastoma (MB), and exposure of newborn mice to ionizing radiation dramatically increases the frequency and shortens the latency of MB. In Ptc1(+/-) mice, MB is characterized by loss of the normal remaining Ptc1 allele, suggesting that genome rearrangements may be key events in MB development. Recent evidence indicates that brain tumors may be linked to defects in DNA-damage repair processes, as various combinations of targeted deletions in genes controlling cell-cycle checkpoints, apoptosis and DNA repair result in MB in mice. Non-homologous end joining (NHEJ) and homologous recombination (HR) contribute to genome stability, and deficiencies in either pathway predispose to genome rearrangements. To test the role of defective HR or NHEJ in tumorigenesis, control and irradiated Ptc1(+/-) mice with two, one or no functional Rad54 or DNA-protein kinase catalytic subunit (DNA-PKcs) alleles were monitored for MB development. We also examined the effect of Rad54 or DNA-PKcs deletion on the processing of endogenous and radiation-induced double-strand breaks (DSBs) in neural precursors of the developing cerebellum, the cells of origin of MB. We found that, although HR and NHEJ collaborate in protecting cells from DNA damage and apoptosis, they have opposite roles in MB tumorigenesis. In fact, although Rad54 deficiency increased both spontaneous and radiation-induced MB development, DNA-PKcs disruption suppressed MB tumorigenesis. Together, our data provide the first evidence that Rad54-mediated HR in vivo is important for suppressing tumorigenesis by maintaining genomic stability.

Disruption of maternal DNA repair increases sperm-derived chromosomal aberrations.

Male and female germ cells can transmit genetic defects that lead to pregnancy loss, infant mortality, birth defects, and genetic diseases in offspring; however, the parental origins of transmitted defects are not random, with de novo mutations and chromosomal structural aberrations transmitted predominantly by sperm. We tested the hypotheses that paternal mutagenic exposure during late spermatogenesis can induce damage that persists in the fertilizing sperm and that the risk of embryos with paternally transmitted chromosomal aberrations depends on the efficiency of maternal DNA repair during the first cycle after fertilization. We show that female mice with defective DNA double-strand break repair had significantly increased frequencies of zygotes with sperm-derived chromosomal aberrations after matings with wild-type males irradiated 7 days earlier with 4 Gy of ionizing radiation. These findings demonstrate that mutagenic exposures during late spermatogenesis can induce damage that persists for at least 7 days in the fertilizing sperm and that maternal genotype plays a major role in determining the risks for pregnancy loss and frequencies of offspring with chromosomal defects of paternal origin.

The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells.

The Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Ercc1-Xpf incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1-Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespective of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for the mechanism of gene targeting in mammalian cells and define a new role for Ercc1-Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1-Xpf in making the recipient genomic locus receptive for gene replacement.

Homologous and non-homologous recombination differentially affect DNA damage repair in mice.

Ionizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the ability of cells to repair the inflicted DNA damage. Here we demonstrate that homologous recombination-deficient mRAD54(-/-) mice are hypersensitive to ionizing radiation at the embryonic but, unexpectedly, not at the adult stage. However, at the adult stage mRAD54 deficiency dramatically aggravates the ionizing radiation sensitivity of severe combined immune deficiency (scid) mice that are impaired in DNA double-strand break repair through DNA end-joining. In contrast, regardless of developmental stage, mRAD54(-/-) mice are hypersensitive to the interstrand DNA crosslinking compound mitomycin C. These results demonstrate that the two major DNA double-strand break repair pathways in mammals have overlapping as well as specialized roles, and that the relative contribution of these pathways towards repair of ionizing radiation-induced DNA damage changes during development of the animal.

Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci formation.

Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4].

The human RAD54 recombinational DNA repair protein is a double-stranded DNA-dependent ATPase.

DNA double-strand break repair through the RAD52 homologous recombination pathway in the yeast Saccharomyces cerevisiae requires, among others, the RAD51, RAD52, and RAD54 genes. The biological importance of homologous recombination is underscored by the conservation of the RAD52 pathway from fungi to humans. The critical roles of the RAD52 group proteins in the early steps of recombination, the search for DNA homology and strand exchange, are now becoming apparent. Here, we report the purification of the human Rad54 protein. We showed that human Rad54 has ATPase activity that is absolutely dependent on double-stranded DNA. Unexpectedly, the ATPase activity appeared not absolutely required for the DNA repair function of human Rad54 in vivo. Despite the presence of amino acid sequence motifs that are conserved in a large family of DNA helicases, no helicase activity of human Rad54 was observed on a variety of different DNA substrates. Possible functions of human Rad54 in homologous recombination that couple the energy gained from ATP hydrolysis to translocation along DNA, rather than disruption of base pairing, are discussed.

Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice.

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vlambda1 light chain genes from naive and memory B cells in DNA repair-deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand-break repair, and AAG-mediated base excision repair.

Disruption of mouse RAD54 reduces ionizing radiation resistance and homologous recombination.

Double-strand DNA break (DSB) repair by homologous recombination occurs through the RAD52 pathway in Saccharomyces cerevisiae. Its biological importance is underscored by the conservation of many RAD52 pathway genes, including RAD54, from fungi to humans. We have analyzed the phenotype of mouse RAD54-/- (mRAD54-/-) cells. Consistent with a DSB repair defect, these cells are sensitive to ionizing radiation, mitomycin C, and methyl methanesulfonate, but not to ultraviolet light. Gene targeting experiments demonstrate that homologous recombination in mRAD54-/- cells is reduced compared to wild-type cells. These results imply that, besides DNA end-joining mediated by DNA-dependent protein kinase, homologous recombination contributes to the repair of DSBs in mammalian cells. Furthermore, we show that mRAD54-/- mice are viable and exhibit apparently normal V(D)J and immunoglobulin class-switch recombination. Thus, mRAD54 is not required for the recombination processes that generate functional immunoglobulin and T cell receptor genes.

Human and mouse homologs of the Saccharomyces cerevisiae RAD54 DNA repair gene: evidence for functional conservation.

BACKGROUND: Homologous recombination is of eminent importance both in germ cells, to generate genetic diversity during meiosis, and in somatic cells, to safeguard DNA from genotoxic damage. The genetically well-defined RAD52 pathway is required for these processes in the yeast Saccharomyces cerevisiae. Genes similar to those in the RAD52 group have been identified in mammals. It is not known whether this conservation of primary sequence extends to conservation of function. RESULTS: Here we report the isolation of cDNAs encoding a human and a mouse homolog of RAD54. The human (hHR54) and mouse (mHR54) proteins were 48% identical to Rad54 and belonged to the SNF2/SW12 family, which is characterized by amino-acid motifs found in DNA-dependent ATPases. The hHR54 gene was mapped to chromosome 1p32, and the hHR54 protein was located in the nucleus. We found that the levels of hHR54 mRNA increased in late G1 phase, as has been found for RAD54 mRNA. The level of mHR54 mRNA was elevated in organs of germ cell and lymphoid development and increased mHR54 expression correlated with the meiotic phase of spermatogenesis. The hHR54 cDNA could partially complement the methyl methanesulfonate-sensitive phenotype of S. cerevisiae rad54 delta cells. CONCLUSIONS: The tissue-specific expression of mHR54 is consistent with a role for the gene in recombination. The complementation experiments show that the DNA repair function of Rad54 is conserved from yeast to humans. Our findings underscore the fundamental importance of DNA repair pathways: even though they are complex and involve multiple proteins, they seem to be functionally conserved throughout the eukaryotic kingdom.

Enrichment of Penicillium chrysogenum microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy.

A procedure to enrich microbodies from Penicillium chrysogenum and a method to evaluate the purity and integrity of the microbodies are described. As a P. chrysogenum microbody marker acyltransferase (AT) was used. The P. chrysogenum hyphae were converted into protoplasts with Novozym 234. In Percoll-sucrose buffer the protoplasts were separated from mycelial debris after 10,000 x g centrifugation. Purified protoplasts were lysed, and the cell homogenate was centrifuged to form a 14,000 x g pellet. After 2 h, 45,000 x g isopycnic centrifugation of the 14,000 x g pellet on a continuous 20-60% nycodenz gradient, ten fractions were collected. The fractions were analyzed for AT containing microbodies by immuno-blotting and immuno-electron microscopy. The results showed that AT-microbodies are enriched in the 38% nycodenz fraction. The microbodies had a diameter of 400 to 500 nm, revealed an intact single membrane and confined AT. The estimated equilibrium density of the P. chyrsogenum microbodies was 1.20 g ml-1 as deduced from the 38% (w/v) nycodenz concentration.

A new rat repetitive DNA family shows preferential localization on chromosome 3, 12 and Y after fluorescence in situ hybridization and contains a subfamily which is Y chromosome specific.

A DNA segment, containing a so far unknown repetitive DNA sequence has been isolated by reassociation of sheared total rat genomic DNA. FISH with this probe gave a strong hybridization signal on the satellites and in the centromeric region of chromosomes 3 and 12 and on the q-arm of the Y chromosome. A much weaker signal was seen in the centromeric region of chromosomes 11, 19 and X. The repeat unit of this repetitive DNA sequence is a 195-200 bp monomer, which is tandemly repeated. Screening of a rat genomic lambda library resulted in the isolation of variant members of this repeat family. FISH results with these members showed differences in their hybridization pattern especially when posthybridization washings were performed under higher stringency. Under these conditions one phage gave a strong hybridization signal only on the Y chromosome; other phage showed only weak hybridization patterns on different chromosomes. A subclone of the Y-specific phage was sequenced and showed large sequence homology with the 195-200 bp monomer. In Southern blot experiments this Y-specific sequence detects several male specific sequences, although some cross-hybridization with closely related sequences in both male and female DNA can also be observed.

Statistical handling of drop-outs in longitudinal clinical trials.

This paper considers the statistical complexities that arise due to outcome related drop-outs in longitudinal clinical trials of the randomized parallel groups design with fixed assessment times and an explanatory aim. The shortcomings of currently popular methods of coping with the problem of drop-outs are discussed. It is proposed that progress can be made by applying the modern methodology that was primarily developed for sample surveys with non-response and for observational studies. A practical application using the Hamilton Rating Scale for Depression is presented.