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Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo.

The C. elegans PAR proteins PAR-3, PAR-6, and PKC-3 are asymmetrically localized and have essential roles in cell polarity. We show that the one-cell C. elegans embryo contains a dynamic and contractile actomyosin network that appears to be destabilized near the point of sperm entry. This asymmetry initiates a flow of cortical nonmuscle myosin (NMY-2) and F-actin toward the opposite, future anterior, pole. PAR-3, PAR-6, and PKC-3, as well as non-PAR proteins that associate with the cytoskeleton, appear to be transported to the anterior by this cortical flow. In turn, PAR-3, PAR-6, and PKC-3 modulate cortical actomyosin dynamics and promote cortical flow. PAR-2, which localizes to the posterior cortex, inhibits NMY-2 from accumulating at the posterior cortex during flow, thus maintaining asymmetry by preventing inappropriate, posterior-directed flows. Similar actomyosin flows accompany the establishment of PAR asymmetries that form after the one-cell stage, suggesting that actomyosin-mediated cortical flows have a general role in PAR asymmetry.

Actins↗

Three multigene families in Plasmodium parasites: facts and questions.

Multigene families optimise fitness by providing a set of related genes with possibly different temporal and/or topological expression patterns. We analyse here the structural organisation and sequence diversity of the rDNA, sera and var C Plasmodium falciparum families, and discuss their consequences for parasite biology. The low rDNA copy number, which reduces reshuffling, is probably the corollary of the need for functionally distinct rRNAs in the insect and in the vertebrate host. The unusual intra-genome and population rDNA sequence diversity results in cells equipped with mosaic ribosome sets. The functional constraints are such that ribosome compatibility could influence parasite fitness and contribute to population structuring. Unlike the dispersed rDNA units, the sera family is arranged as a tandem gene cluster, with seven contiguous similar genes, and one more distantly related paralog. We address the question of the inclusion criteria in family definition. We discuss the results concerning the SERA proteins expression and function in the context of the long overlooked multigene family. The var C module is shared by var genes, 'orphan' var C and var C pseudogenes. Analysis of 125 var C deduced protein sequences highlights a well-conserved framework, including putative phosphorylation sites, consistent with the proposed function of mediating interaction with cytoskeletal proteins. The 5' and 3' flanking sequences of the var C pseudogenes are heterogeneous. In contrast, the flanking sequences of the uninterrupted var C modules show remarkable conservation. This is interesting in view of the silencing activity of the var intronic sequence on var expression. The 5' flanking sequence dichotomy reported for internal and sub-telomeric var genes extends to the 3' flanking sequences. This has profound implications for transcription regulation and generation of diversity. The var C family suggests a role for pseudogenes as a diversity reservoir and in genome dynamics by promoting ectopic recombination.

Amino Acid Sequence↗

Pivotal role of Gly 121 in dihydrofolate reductase from Escherichia coli: the altered structure of a mutant enzyme may form the basis of its diminished catalytic performance.

The structure and folding of dihydrofolate reductase (DHFR) from Escherichia coli and the mutant G121V-DHFR, in which glycine 121 in the exterior FG loop was replaced with valine, were studied by molecular dynamics simulations and CD and fluorescence spectroscopy. The importance of residue 121 for the chemical step during DHFR catalysis had been demonstrated previously. High-temperature MD simulations indicated that while DHFR and G121V-DHFR followed similar unfolding pathways, the strong contacts between the M20 loop and the FG loop in DHFR were less stable in the mutant. These contacts have been proposed to be involved in a coupled network of interactions that influence the protein dynamics and promote catalysis [Benkovic, S. J., and Hammes-Schiffer, S. (2003) Science 301, 1196-1202]. CD spectroscopy of DHFR and G121V-DHFR indicated that the two proteins existed in different conformations at room temperature. While the thermally induced unfolding of DHFR was highly cooperative with a midpoint at 51.6 +/- 0.7 degrees C, G121V-DHFR exhibited a gradual decrease in its level of secondary structure without a clear melting temperature. Temperature-induced unfolding and renaturation from the urea-denatured state revealed that both proteins folded via highly fluorescent intermediates. The formation of these intermediates occurred with relaxation times of 149 +/- 4.5 and 256 +/- 13 ms for DHFR and G121V-DHFR, respectively. The fluorescence intensity for the intermediates formed during refolding of G121V-DHFR was approximately twice that of the wild-type. While the fluorescence intensity then slowly decayed for DHFR toward a state representing the native protein, G121V-DHFR appeared to be trapped in a highly fluorescent state. These results suggest that the reduced catalytic activity of G121V-DHFR is the consequence of nonlocal structural effects that may result in a perturbation of the network of promoting motions.

Catalysis↗

Tau induces cooperative Taxol binding to microtubules.

Taxol and tau are two ligands that stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds beta tubulin in the MT interior. Tau is a MT-associated protein that binds both alpha and beta tubulin on the MT exterior. Both Taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts to bundle, stiffen, and space MTs. A structural study recently suggested that Taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces Taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau, yields the equilibrium dissociation constant of approximately 2 microM, and determines the escape rate of Taxol through one pore to be 1.7 x 10(3) (M x s)(-1). Extension of the model yields a measure of Taxol cooperativity with a Hill coefficient of at least 15 when tau is present at a 1:1 molar ratio with tubulin.

Animals↗

Dynamic regulation of cyclooxygenase-2 promoter activity by isoforms of CCAAT/enhancer-binding proteins.

To elucidate the mechanism by which isoforms of CCAAT/enhancer-binding proteins regulate cyclooxygenase-2 expression, we determined by a novel technique binding of six isoforms of this transactivator to two sequence-specific CCAAT/enhancer-binding protein (-132/-125) and cyclic AMP (-59/-53) regulatory elements in human foreskin fibroblasts treated with phorbol 12-myristate 13-acetate for 4 h. The delta isoform bound to these two elements at basal state, which was displaced by full-length as well as two truncated beta isoforms, a 41-kDa liver-enriched activating protein and a 16-kDa liver-enriched inhibitory protein, after phorbol ester stimulation. Kinetic analysis shows time-dependent changes in beta and delta binding that were concordant with time-dependent increase in cyclooxygenase-2 induction. Overexpression of the 16-kDa beta isoform blocked the promoter activity and protein level induced by phorbol ester. Paradoxically, it increased binding of beta isoforms to the sequence-specific promoter DNA but suppressed cyclooxygenase-2 promoter activation by p300 cotransfection. These findings provide new insight into the regulation of cyclooxygenase-2 promoter by an interplay between two opposite beta isoforms and p300 co-activator.

Acetyltransferases↗

SGEF, a RhoG guanine nucleotide exchange factor that stimulates macropinocytosis.

SGEF (SH3-containing Guanine Nucleotide Exchange Factor) is a RhoGEF of unknown function. We found the SGEF protein to be expressed in many established cell lines and highly expressed in human liver tissue. SGEF stimulated the formation of large interconnected membrane ruffles across dorsal surfaces when expressed in fibroblasts. SGEF required its proline-rich amino-terminus to generate dorsal, but not lateral, membrane ruffles and a functional SH3 domain to colocalize with filamentous actin at sites of membrane protrusion. Full-length SGEF activated RhoG, but not Rac, when expressed in fibroblasts. Further, recombinant SGEF DH/PH protein exchanged nucleotide on RhoG, but not on Rac1 or Rac3, in vitro. Scanning electron microscopy of fibroblasts demonstrated that SGEF induced dorsal ruffles that were morphologically similar to those generated by constitutively active RhoG, but not constitutively active Rac1. Transient expression of SGEF stimulated fibroblast uptake of 10-kDa dextran, a marker of macropinocytosis. This required the full-length protein and a catalytically active DH domain. Finally, activated RhoG was found to be more effective than activated Rac, and comparable to SGEF, in its ability to trigger dextran uptake. Together, this work establishes SGEF as a RhoG exchange factor and provides evidence that both SGEF and RhoG regulate membrane dynamics in promotion of macropinocytosis.

Actins↗

CLIP-170 homologue and NUDE play overlapping roles in NUDF localization in Aspergillus nidulans.

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32 degrees C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150(Glued) dynactin, and NUDF were all seen as plus-end comets at 32 degrees C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32 degrees C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42 degrees C).

Amino Acid Sequence↗

The evolution of transcriptional regulation in eukaryotes.

Gene expression is central to the genotype-phenotype relationship in all organisms, and it is an important component of the genetic basis for evolutionary change in diverse aspects of phenotype. However, the evolution of transcriptional regulation remains understudied and poorly understood. Here we review the evolutionary dynamics of promoter, or cis-regulatory, sequences and the evolutionary mechanisms that shape them. Existing evidence indicates that populations harbor extensive genetic variation in promoter sequences, that a substantial fraction of this variation has consequences for both biochemical and organismal phenotype, and that some of this functional variation is sorted by selection. As with protein-coding sequences, rates and patterns of promoter sequence evolution differ considerably among loci and among clades for reasons that are not well understood. Studying the evolution of transcriptional regulation poses empirical and conceptual challenges beyond those typically encountered in analyses of coding sequence evolution: promoter organization is much less regular than that of coding sequences, and sequences required for the transcription of each locus reside at multiple other loci in the genome. Because of the strong context-dependence of transcriptional regulation, sequence inspection alone provides limited information about promoter function. Understanding the functional consequences of sequence differences among promoters generally requires biochemical and in vivo functional assays. Despite these challenges, important insights have already been gained into the evolution of transcriptional regulation, and the pace of discovery is accelerating.

Animals↗

The Family to Family program: a structural intervention with implications for the prevention of HIV/AIDS and other community epidemics.

OBJECTIVES: To describe a case study of a community-based intervention located in the Harlem community of New York City, one of the oldest African-American communities in the United States. Although not specifically designed to prevent HIV infection, the program, 'Family to Family', exemplifies a 'structural intervention' that was created to strengthen family functioning and to strengthen the bonds that connect families to each other. By fostering strong relationships within and between families in a community with high rates of violence, drug abuse, and HIV infection, the program seeks to improve the quality of neighborhood life and influence the social determinants of individual risk behavior. SOCIAL CAPITAL: Family to Family was created specifically to develop the 'social capital' that is available to children and families in the Harlem community. Social capital refers to resources that result from social relationships, and that enhance an individual's or a group's ability to function and achieve a given set of goals and objectives. In addition to fostering closer relationships between children and their parents, this program also works to help participating families develop closer relationships with other participating families and with Columbia University student and faculty volunteers. Finally, Family to Family is sustained through the efforts of volunteers; it receives no grant support and is entirely self-supporting. CONCLUSIONS: Family to Family has the potential to change the social dynamics that promote HIV risk behavior in communities such as Harlem. Should it prove successful in improving the relationships between families and children, and in increasing the social capital available to all of its participants, it may become an important asset to public health prevention specialists concerned about preventing the spread of HIV.

Acquired Immunodeficiency Syndrome↗

Role of resistance training in heart disease.

Since the mid-1980s resistance training has become an accepted part of the exercise rehabilitation process for patients eligible for traditional cardiac rehabilitation programs. A growing number of studies have demonstrated the safety of resistance training in Phase III/IV programs (Phase III--community based, beginning 6-12 wk posthospital discharge; a typical patient would be clinically stable with a functional capacity of > or = 5 METs; Phase IV--long-term maintenance) and more recently in Phase II (beginning within 3 wk posthospital discharge and lasting up to 3 months). Evidence is consistent that this form of training provokes fewer signs and symptoms of myocardial ischemia than aerobic testing and training, perhaps because of a lower heart rate (HR) and higher diastolic pressure combining to produce improved coronary artery filling. The major role of resistance training in heart disease patients is to promote increased dynamic muscle strength. Increases in muscular strength have been associated with increased peak exercise performance, improved submaximal endurance, and reduced ratings of perceived leg effort. Two studies show that resistance training may result in improved self-efficacy for strength and exercise tasks and improved quality of life parameters such as total mood disturbance, depression/dejection, fatigue/inertia, and emotional health domain scores. The data on risk factor modification are somewhat equivocal. Studies on blood lipid profiles have mostly been contaminated by confounders, and the effects on blood pressure (BP) are inconsistent. There are encouraging reports that resistance training may increase glucose tolerance and insulin sensitivity, independent of changes in body fat or aerobic capacity. Future studies are needed in patients with congestive heart failure and orthotopic heart transplantation; muscle weakness is common in these groups and makes them excellent candidates to benefit from this form of exercise.

Blood Pressure↗

Enhancing presentation skills for the advanced practice nurse: strategies for success.

Professional speaking is a component of the professional practice role of the advanced practice nurse (APN). The skills to communicate effectively to one person or an audience of 100 provide the APN with the essential tools for implementing change, collaborating effectively, presenting information at professional meetings, or communicating the impact of clinical outcomes in the boardroom. Public speaking skills, a professional image, and improved communication can facilitate advancement along any career ladder. The greater your fear, the more self-confidence you will gain by stepping up to a challenge and conquering it. This article describes strategies for organizing and presenting your message in a clear and concise format. Techniques to manage the anxiety produced when attempting to articulate your thoughts is essential for effective communication. Skills for enhancing the delivery of your message through effective body language, professional image, voice modulation, and use of audiovisual aids are addressed. Creative techniques for fielding questions are key in promoting a dynamic closure and provide consistent reinforcement of the key message content.

Attitude of Health Personnel↗

GPER stimulation attenuates mitochondrial dysfunction and cardiac dysfunction in ovariectomized mice with heart failure with preserved ejection fraction (HFpEF).

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is prevalent among postmenopausal women and is strongly linked to estrogen deficiency. G-protein coupled estrogen receptor (GPER) mediates non-genomic estrogen signalling and exerts cardiovascular protective effects. Its role in the pathogenesis of HFpEF remains unclear. This study aimed to explore whether GPER activation could attenuate mitochondrial dysfunction and cardiac damage in ovariectomized (OVX) mice with HFpEF. METHODS: Circulating GPER levels were measured in postmenopausal women with HFpEF and healthy controls. A correlation analysis was performed to assess the associations between GPER and cardiac function. Female C57BL/6J mice underwent ovariectomy and were fed with high-fat diet and l-NAME to induce HFpEF. Mice were treated with the GPER agonist G-1 for 4 weeks. Cardiac function, histological changes, oxidative stress, mitochondrial function and mitophagy were evaluated in vivo and in vitro. RESULTS: Serum GPER levels were significantly higher in postmenopausal women with HFpEF and correlated with NT-proBNP and E/e'. In OVX mice with HFpEF, GPER expression was up-regulated, and G-1 improved diastolic function, reduced myocardial hypertrophy and oxidative stress. Importantly, G-1 restored mitochondrial ATP production, normalized mitochondrial dynamics and promoted mitophagy in vivo and in vitro. These effects were associated with activation of the AMPK/ULK1 pathway. Inhibition of AMPK diminished the protective effects of G-1 in cardiomyocytes. CONCLUSIONS: GPER agonist G-1 ameliorated mitochondrial dysfunction, promoted mitophagy and alleviated cardiac diastolic dysfunction in OVX mice with HFpEF, partially through the AMPK/ULK1 pathway, indicating GPER as a therapeutic target for postmenopausal women with HFpEF.

AMPK/ULK1 signalling pathway↗

Administration of medicines by emergency nurse practitioners according to protocols in an accident and emergency department.

OBJECTIVE: To present the legal and professional issues related to nurse administration of drugs according to protocols, and describe the implementation and initial audit findings of such a scheme. SETTING: Accident and emergency (A&E) department of a district general hospital. METHODS: Analysis of legal and professional opinion. Protocols acceptable to the medical, nursing, and pharmacy professions were developed across a wide range of drugs appropriate for administration by accident and emergency nurse practitioners (ENPs). The first six months of the scheme were audited. Audit initially addressed general compliance with protocols and later the specific areas of tetanus immunisation and emergency contraception. RESULTS: ENPs assessed 2925 patients in six months (10.9% of all new patients); 455 patients (15.5% of the ENP patients) were given drugs according to protocols. There were no breaches of the protocols. Subsequent audit of tetanus immunisation showed 94-100% compliance with protocol standards and 71-100% compliance for emergency contraception. CONCLUSIONS: There are no legal or professional obstacles to the development of protocols for the administration of drugs to patients by nurses without reference to a doctor, providing the protocols meet all the requirements of the UKCC and have the support of consultant medical staff. Such a system must be subject to regular audit to promote a dynamic approach to protocols and training. The system safely enhanced the quality of care of patients treated by ENPs in A&E.

Clinical Protocols↗

Matrix expression and proliferation of primary gingival fibroblasts in a three-dimensional cell culture model.

The growth of cultured primary human gingival fibroblasts and the three-dimensional arrangement of the extracellular matrix in a polyester carrier system was investigated using various histological techniques. The results were compared with monolayer cultures. Collagen types I, III, V, and VI were investigated by conventional and fluorescence microscopy, scanning and transmission electron microscopy, and confocal laser scanning microscopy. Human gingival fibroblasts were obtained from tissue biopsies of five donors and were cultivated up to 5 weeks under three-dimensional culture conditions. The cells displayed an elongated, spindle-like or stellate morphology resembling the in vivo situation. Collagen type I revealed thick fiber bundles, and collagens type III and V were distributed as fine fibrils or small bundles throughout the culture system. Frequently, the fibers were oriented parallel to the long axis of the cells. Type VI collagen formed thin fibers and revealed a reticular pattern. In histological sections the cultured cells exhibited a morphology clearly different from that of cells cultured in monolayers. Their shape and spatial distribution resembled that of cells in tissue biopsies more closely. The culture system presented here promotes a dynamic model for performing studies for instance on the interactions of cultured cells with extracellular matrix molecules, on the pathogenesis of inflammatory processes or on the interactions with biomaterials, thus providing qualitative and quantitative information.

Adolescent↗

Helicobacter pylori and gastric cancer.

Gastric cancer despite a declining incidence remains a significant cause of morbidity and mortality world wide. There is strong epidemiological and histological evidence to associate Helicobacter pylori infection with the subsequent development of gastric cancer. The exact pathophysiological mechanisms involved remain to be elucidated. There is evidence to relate Helicobacter pylori infection and subsequent inflammation with an increase in gastric epithelial cell proliferation and with the induction of apoptosis. Such alterations in cellular dynamics may promote the development of mitogenic cell lines by inducing DNA damage. Studies have shown that following successful treatment, proliferation rates return to normal. At what histological stage, eradication is of benefit is less clear. It is likely that following the development of atrophy or intestinal metaplasia eradication will only slow progression. It would, therefore, seem logical, that to establish any benefit for a population, treatment should be employed at an earlier stage. As yet, an at risk group has not been identified, and as such population screening cannot be advised, mainly as a result of financial implications and the risk of promoting the development of resistant strains. Recent studies have explored the rules of bacterial factors, CagA and VacA status, host factors, HLA type, and environmental factors as determinants of outcome. Results have been variable. The establishment of an at risk group would enable selective screening and treatment, and thus prevent the development of gastric carcinoma as a result of Helicobacter pylori infection in the long-term.

Adenocarcinoma↗

[On the improvement of everyday life behavior of chronic schizophrenic patients; from the viewpoint of "play-like behavior"].

In this study, the author examined the behavioral patterns of chronic schizophrenic inpatients to follow the process of the amelioration of abulic symptoms such as loss of interest, poverty of thought, lack of sociality, and poor communication. In everyday life in the ward, abulic patients had difficulties in accomplishing not only the basic habitual acts such as getting up or going to bed regularly, exchanging greetings, cleaning teeth, bathing, and washing clothes, but also their assigned duties on the ward. Furthermore, they were unable to behave according to the rules for inpatients, express their emotions appropriately, or build normal interpersonal relationship. The author found that five inpatients achieved some spontaneous behaviors of their own choices in the process of improvement in the above-mentioned habitual acts. As these spontaneous behaviors proceeded through several phases, obvious improvements in their behavioral patterns in everyday life were also observed. The initial phase of transient spontaneous behavior was followed by the second phase of continual spontaneous behavior. Finally, in the interview sessions, the patients became to express pleasurable emotions and physical feelings when they performed their own acts of continual spontaneous behavior. This phenomenon seemed valuable in the therapeutic context because schizophrenic patients are considered seldom capable of having positive feelings toward their own experiences. Therefore, these pleasurable continual spontaneous behaviors may be called "play-like behavior", as confirmed by comparison with the properties of "play" as defined by Caillois. In considering schizophrenic autism, Minkowski described "activité autiste" as an intrinsic quality of the way of life in schizophrenic patients. The manifestation of such quality in spontaneous behaviors can be regarded as having two meanings; an aspect of pathological acting out and a sign of recovery to realistic behaviors. Therapists should consider both aspects when conducting therapies. Although the patients regained the habits and norms of everyday life during hospitalization, the rules involved in "play-like" behavior seemed to contradict some rules in the habitual acts or assigned duties in their daily lives because within "play-like" behavior, freedom predominates over rules. The rules in "play-like" behavior are acquired mostly by mimicking other people. These rules are not fixed laws with penalties but are changeable rules depending on the circumstances of the behavioral process. From this viewpoint, "play-like" behavior allows the patients to acquire practical rules and to understand the relative nature of the rules. Most of the "play-like" behaviors originate from the preferences of the individual patients, which may be something that they have already experienced, something that they have longed for, or something that gives them self-fulfillment. These qualities contribute to the acquisition of experiences that they find pleasurable. For the patients, recalling each "play-like" behavior in the interview sessions enables them to grasp the whole picture of their behaviors, to reinforce their attachment to these behaviors as their own experiences, and to promote the dynamic process between behavior and thought. In this sense, psychotherapy incorporating the aspect of "play-like" behavior seems to prepare the schizophrenic patients to improve their way of thinking. To nurture "play-like" behavior helps the chronic schizophrenic patients to recover will power, independence and sociality, and contributes to the improvement of clinical symptoms and of daily life activities.

Adult↗

Bibliotherapy: the therapeutic use of didactic and literary texts in treatment, diagnosis, prevention, and training.

The term bibliotherapy has been defined by Russell and Shrodes as "a process of dynamic interaction between the personality of the reader and literature--an interaction which may be used for personality assessment, adjustment, and growth." In the clinical setting, the dynamics that promote change in a patient-reader can include identification, projection, introjection, catharsis, and insight. Clinicians may use bibliotherapy as a tool for patient treatment, medical diagnosis, and the prevention of illness related to psychosocial dysfunction, allowing for gradual and mutual insight into patient complaints over time. Bibliotherapy may display efficacy on intellectual, psychosocial, interpersonal, emotional, and behavioral levels. The author identifies two basic types of resources that are useful to clinicians administering bibliotherapy: didactic texts, which are instructive, and imaginative literature, which can be a literary text, biography, or autobiography and fosters an imaginative response from the patient-reader. The author identifies the advantages and risks of using bibliotherapy and explores its possible applications in osteopathic medical education, encouraging osteopathic medical educators to familiarize themselves with this treatment modality.

Bibliotherapy↗