*Article* **SspH, a Novel HATPase Family Regulator, Controls Antibiotic Biosynthesis in** *Streptomyces*

**Xue Yang 1,†, Yanyan Zhang 1,\* ,†, Shanshan Li 1 , Lan Ye 1,2, Xiangjing Wang 2 and Wensheng Xiang 1,2,\***

† These authors contributed equally to this work.

**Abstract:** *Streptomyces* can produce a wealth of pharmaceutically valuable antibiotics and other bioactive compounds. Production of most antibiotics is generally low due to the rigorously controlled regulatory networks, in which global/pleiotropic and cluster-situated regulatory proteins coordinate with various intra- and extracellular signals. Thus, mining new antibiotic regulatory proteins, particularly the ones that are widespread, is essential for understanding the regulation of antibiotic biosynthesis. Here, in the biopesticide milbemycin producing strain *Streptomyces bingchenggensis*, a novel global/pleiotropic regulatory protein, SspH, a single domain protein containing only the HATPase domain, was identified as being involved in controlling antibiotic biosynthesis. The *sspH* overexpression inhibited milbemycin production by repressing the expression of milbemycin biosynthetic genes. The *sspH* overexpression also differentially influenced the expression of various antibiotic biosynthetic core genes. Site-directed mutagenesis revealed that the HATPase domain was essential for SspH's function, and mutation of the conserved amino acid residues N54A and D84A led to the loss of SspH function. Moreover, cross-overexpression experiments showed that SspH and its orthologs, SCO1241 from *Streptomyces coelicolor* and SAVERM\_07097 from *Streptomyces avermitilis*, shared identical functionality, and all exerted a positive effect on actinorhodin production but a negative effect on avermectin production, indicating that SspH-mediated differential control of antibiotic biosynthesis may be widespread in *Streptomyces*. This study extended our understanding of the regulatory network of antibiotic biosynthesis and provided effective targets for future antibiotic discovery and overproduction.

**Keywords:** global regulator; milbemycins; *Streptomyces bingchenggensis*; SspH; actinorhodin; avermectin


**5. MW dielectrics with "mobile sublattice"**

The authors of [29] reported the development of novel MW dielectrics with high temperature stability of dielectric parameters based on solid solutions, where one of the phases (lithiumcontaining La1/2Li1/2TiO3) had a positive temperature coefficient of permittivity (TCε > 0). However, there was no explanation of the nature of this fact in literature.

The phase La1/2Li1/2TiO3, which crystallizes in perovskite structure, belongs to Ln2.3-xM3xTio3 solid solutions (Ln = rare-earth elements, M = alkali metal ion) (Fig 6). In this system, the M ions partially substitute for rare-earth ions. In this case, the electroneutrality condition is satisfied [30, 31]. If M = Na, K, materials crystallize in perovskite structure and are characterized by low dielectric loss (tg δ ≤ 10-3) and high permittivity in the MW range (about 100) and TCε < 0 [32].

At the same time, when M = Li, a system of solid solutions is formed, in which rare-earth ions, lithium ions, structural vacancies, which are characterized by TCε > 0, are in one sublattice at the same time. Lithium ions can move along structural channels, ensuring a high lithium-ion conductivity [33, 34], positive temperature coefficient of permittivity (TCε > 0) and causing considerable dielectric loss in the MW range. The latter is inadmissible in the creation of high-Q dielectrics. Investigations showed, however, that the dielectric loss in the MW range can be greatly reduced by decreasing lithium ion conductivity. The latter is achieved by substituting rare-earth ions with smaller radius for lanthanum ions. This leads to a decrease in the size of structural channels, in which lithium ions are; this reduces dielectric loss in the MW range through a decrease in lithium ion mobility in the structure. In this case, TCε > 0 is retained. As a result, solid solutions have been obtained, in which rare-earth ions are simultaneously substituted by alkali metal ions with large (Na, K) and small (Li) radius and which have a high temperature stability of dielectric parameters in the MW range (Table 2) [35].

**Figure 6.** Perovskite structure of La2/3-xLi 3xTiO3

120 Dielectric Material

transitions [22], which was confirmed by the authors of [24], who carried out synchrotron Xray diffraction studies of Ba4.5Sm9Ti18O54 samples in the temperature range 10-295 K. These data indicate the absence of structural transitions in the temperature ranges where anomalies of dielectric parameters were observed. It can be concluded that the temperature dependence anomalies of dielectric parameters are not coupled with the peculiarities of

On the basis of an analysis it was assumed that the nature of the anomaly of dielectric parameters is coupled with harmonic and anharmonic BLTss lattice vibration, which is different in the character of influence on the temperature behavior of dielectric parameters [23]. Investigations showed that the plots of ε and tg δ against temperature depend largely upon harmonic and anharmonic lattice vibration modes. Therefore, using different hetero- and isovalent substitutions in cation sublattices, one can influence the lattice phonon spectrum and hence obtain materials with high temperature stability of dielectric parameters, which are used

The authors of [29] reported the development of novel MW dielectrics with high temperature stability of dielectric parameters based on solid solutions, where one of the phases (lithiumcontaining La1/2Li1/2TiO3) had a positive temperature coefficient of permittivity (TCε > 0).

The phase La1/2Li1/2TiO3, which crystallizes in perovskite structure, belongs to Ln2.3-xM3xTio3 solid solutions (Ln = rare-earth elements, M = alkali metal ion) (Fig 6). In this system, the M ions partially substitute for rare-earth ions. In this case, the electroneutrality condition is satisfied [30, 31]. If M = Na, K, materials crystallize in perovskite structure and are characterized by low dielectric loss (tg δ ≤ 10-3) and high permittivity in the MW range

At the same time, when M = Li, a system of solid solutions is formed, in which rare-earth ions, lithium ions, structural vacancies, which are characterized by TCε > 0, are in one sublattice at the same time. Lithium ions can move along structural channels, ensuring a high lithium-ion conductivity [33, 34], positive temperature coefficient of permittivity (TCε > 0) and causing considerable dielectric loss in the MW range. The latter is inadmissible in the creation of high-Q dielectrics. Investigations showed, however, that the dielectric loss in the MW range can be greatly reduced by decreasing lithium ion conductivity. The latter is achieved by substituting rare-earth ions with smaller radius for lanthanum ions. This leads to a decrease in the size of structural channels, in which lithium ions are; this reduces dielectric loss in the MW range through a decrease in lithium ion mobility in the structure. In this case, TCε > 0 is retained. As a result, solid solutions have been obtained, in which rare-earth ions are simultaneously substituted by alkali metal ions with large (Na, K) and small (Li) radius and which have a high temperature stability of dielectric parameters in the

in modern decimeter and centimeter wave band communication systems [25, , , 28].

However, there was no explanation of the nature of this fact in literature.

sample preparation and the presence of structural transitions.

**5. MW dielectrics with "mobile sublattice"**

(about 100) and TCε < 0 [32].

MW range (Table 2) [35].

**Table 2.** Dielectric parameters of the Sm1/2Li1/2TiO3 – (1-x) Sm1/2Na1/2TiO3 system at 10 GHz


Our findings confirmed our first hypothesis that the AATtraining is suitable for changing HSAs' avoidance of smiling faces. Specifically, HSAs trained to approach smiling faces became faster in pulling smiling faces closer to themselves. After the training, they showed a significant face-approach tendency. Similarly, participants trained to avoid smiling faces showed a significant face-avoidance tendency. Hence, we can argue that the AAT is an appropriate and promising method to train HSAs to approach smiling faces. Moreover, it may have potential therapeutic value in the sense that it could help to reduce *automatic* avoidance behavior in social anxiety, in addition to the existing therapeutic tools for reducing *controlled* avoidance behavior.

Furthermore, our findings partially confirmed our second hypothesis as well. The trained action tendencies of the Pull-Push AAT seemed to generalize, to some extent, to new faces

3We also analyzed the sum score of the positive mood items and the sum score of the negative items separately. This yielded results which mirrored those of the joint analysis reported here.

in a new type of AAT, the Face-Turn-AAT (Voncken et al., 2011). Here, HSAs trained to pull smiling faces closer were faster to turn female faces toward themselves. However, it must be noted that no effect on male faces was observed. An explanation for this finding could be that the female faces were perceived as less threatening by the predominantly female HSAs of the current experiment. Accordingly, future studies should determine if and how the training effects can be extended to male faces in order to increase the effectiveness of the training for treatment purposes. These studies should also pay attention to possible interactions of participant gender, model gender, and emotion because there is evidence that the processing of smiling facial expressions depends on the gender of both the person expressing them and the person perceiving them (LaFrance et al., 2003).

Finally, we expected that when experiencing a social challenge, HSAs who had received approach-smiling-faces training would feel generally better and would experience less anxiety than HSAs who received avoid-smiling-faces training. The reported mood ratings and anxiety ratings were in accordance with this hypothesis. Compared to HSAs of the avoid-faces training, HSAs of the approach-faces training reported both higher positive mood in general, and lower levels of anxiety in particular, after videotaping their self-presentation and expecting it to be evaluated by a peer. Interestingly, there were no differences between the two groups in general mood or specific anxiety when they received instructions for the self-presentation. This suggests that the training affected mood recovery after stress rather than anticipatory fear: HSAs who had been trained to approach smiling faces appeared to *recover* more easily after the social threat task. This result is in accordance with studies that tested the effects of another type of training, attention bias modification, on emotional vulnerability (MacLeod et al., 2002; Amir et al., 2008). As in our study, only participants' anxiety *after* the stressful event was affected by the training condition. Moreover, as in many previous studies (e.g., MacLeod et al., 2002), our training was not a selective mood induction: directly afterwards, both training groups felt slightly worse than before. This is understandable, given the somewhat boring nature of the training.

In the current study, we experimentally manipulated whether the participants were trained to approach or to avoid smiling faces. This way, we can safely conclude that automatic approachavoidance tendencies are indeed causal factors in the behavior and subjective fear of socially anxious individuals. This is important from a theoretical point of view, e.g., for the evaluation of cognitive theories of anxiety. However, it does not yet prove the therapeutic value of an approach training in SAD because the positive approach-smiling faces training was not compared to a placebo training condition. In this respect, our study nicely complements the one recently reported by Taylor and Amir (2012) who found positive effects of an approachfaces training, compared to a placebo training condition. In this study, however, the authors only found effects of the training on social interaction behavior, not on subjective fear. For the latter, several differences between the two studies may be responsible. For instance, our social stress task may have been more threatening, or our training may have been more powerful because it involved the approach of smiling faces rather than neutral faces. Therefore, future studies should compare the approach-smiling faces training to a placebo training condition, preferably in diagnosed social phobics, and study effects on both behavior and social anxiety levels. Moreover, studies with delayed follow-up measurements would be helpful for determining the duration of approach-avoidance training effects.

Several limitations of the current study deserve mentioning. First, the sample was rather small, and it contained more female than male participants, therefore it was impossible to test the participant gender × model gender × emotion interaction mentioned above. Also, we had neither a non-anxious control group nor a group of SAD patients to compare the pre-training AAT scores to. Moreover, the study is lacking emotional expressions other than happy faces. Finally, the exact mechanisms by which the current training reaches its positive effects on stress recovery remain to be determined. We have reason to assume that more than a response bias was induced, given the observed partial transfer to another task and the effects on mood. At least two mechanisms might be at work here. First, operant evaluative conditioning: approaching certain stimuli repeatedly can make these stimuli more pleasant, compared to repeatedly avoiding them (e.g., Woud et al., 2011). This effect seems to be strongest for ambivalent stimuli, which smiling faces are for HSAs. Second, if the smiling faces were experienced as threatening, their repeated presentation might have caused habituation and extinction of the fear reaction, and this effect might have been stronger for those participants who approached the faces without experiencing any negative consequences.

To summarize, we have demonstrated that it is possible to change avoidance tendencies in HSAs using an AAT training procedure, and that the training effects partly generalize to other situations involving automatic approach behavior. Moreover, training HSAs to approach rather than to avoid smiling faces led to less self-reported anxiety after a threatening social task, thereby fostering recovery from stress. These results corroborate the causal role of automatic approach-avoidance tendencies in social anxiety. Moreover, the results suggests that the AAT is not only suitable for the assessment of biased avoidance behavior in social anxiety, but also for the re-training of this automatic behavior. Since avoidance behavior plays an important role in the maintenance of SAD, an effective training to approach smiling faces could be an important step toward the improvement of social interactions in socially anxious individuals. Thus, the approach-smiling-faces training could be a promising addition to more traditional treatments of social anxiety.

## **ACKNOWLEDGMENTS**

This study was supported by the Behavioral Science Institute (BSI) of Radboud University Nijmegen. We are grateful to Carla van Lier for supplying the self-presentation used in the experiment, and to Frank Leonhardt for programming the AAT. Helpful comments by two anonymous reviewers are also gratefully acknowledged.

## **REFERENCES**

Social anxiety predicts avoidance behaviour in virtual encounters. *Cogn. Emot.* 24, 1269–1276. doi: 10.1080/02699930903309268

*Psychol. Sci*. 22, 490–497. doi: 10.1016/j.jbtep.2010.11.004

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 05 April 2013; accepted: 30 July 2013; published online: 20 August 2013. Citation: Rinck M, Telli S, Kampmann IL, Woud ML, Kerstholt M, te Velthuis S, Wittkowski M and Becker ES (2013) Training approach-avoidance of smiling faces affects emotional vulnerability in socially anxious individuals. Front. Hum. Neurosci. 7:481. doi: 10.3389/ fnhum.2013.00481*

*This article was submitted to the journal Frontiers in Human Neuroscience.*

*Copyright © 2013 Rinck, Telli, Kampmann, Woud, Kerstholt, te Velthuis, Wittkowski and Becker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Looking on the bright side in social anxiety: the potential benefit of promoting positive mental imagery

## **Arnaud Pictet\***

Psychiatry, Experimental Psychopathology and Cognitive Therapies, University of Oxford, Geneva, Switzerland

### **Edited by:**

Pierre Philippot, Catholic University of Louvain at Louvain-la-Neuve, Belgium

**Reviewed by:**

Virginie Peschard, University of Louvain, Belgium Jennifer Wild, University of Oxford, UK

#### **\*Correspondence:**

Arnaud Pictet, Psychiatry, Experimental Psychopathology and Cognitive Therapies, University of Oxford, 41A route de Frontenex, 1207 Geneva, Switzerland e-mail<EMAIL_ADDRESS>
Current cognitive models of social phobia converge on the view that negative imagery is a key factor in the development and maintenance of the disorder. Research to date has predominantly focussed on the detrimental impact of negative imagery on cognitive bias and anxiety symptoms, while the potential benefit of promoting positive imagery has been relatively unexplored. Emerging evidence suggests however that positive imagery could have multiple benefits such as improving positive affect, self-esteem and positive interpretation bias, and enhancing social performance. The present article defends the view that combining bias induction with a repeated practice in generating positive imagery in a cognitive bias modification procedure could represent a promising area for future research and clinical innovation in social anxiety disorder.

**Keywords: social anxiety, mental imagery, cognitive bias modification, treatment, combined cognitive bias**

## **NEGATIVE IMAGERY IN SOCIAL ANXIETY**

People with social anxiety disorder (SAD) frequently report experiencing negative self-imagery of how they think they appear to others in feared social situations (Hackmann et al., 2000). The assumption that this negative imagery may be important in maintaining social anxiety is a central tenet of most cognitive models of SAD (Clark and Wells, 1995; Rapee and Heimberg, 1997; Hofmann, 2007). For example, Clark and Wells (1995) model suggests that when socially anxious individuals enter a feared social situation, they shift their attention towards internal cues and use this information to infer how they appear to others. This internal information can often take the form of a distorted, negative self-image that individuals perceive as accurate.

Empirical support for the causal role of negative imagery in social anxiety has been established through a series of experimental studies (Hirsch et al., 2003, 2004; Stopa and Jenkins, 2007). Hirsch and colleagues conducted a series of experiments in which socially anxious participants were asked to hold an image in mind while taking part in two conversations with a stranger. During one conversation they were asked to hold in mind a negative image of the sort they would typically generate in anxiety provoking social situations, whereas during the other conversation they were instructed to hold a less negative (control) image in mind (based on a memory of a social situations in which they felt relaxed). Participants reported feeling more anxious when holding the negative image than the benign one. They also judged their social performance more negatively both in terms of showing more symptoms of anxiety and performing less well during the conversation.

Further support for the causal role of negative imagery in social anxiety has been provided in experimental studies conducted with low anxious participants. In Hirsch et al. (2005), participants who were normally confident public speakers were asked to generate a negative, positive or control (unrelated) selfimage prior to giving a speech. Findings from socially anxious samples were replicated, with participants in the negative imagery condition reporting greater anxiety and judging their social performance as being poorer than those assigned to the positive imagery condition. A further line of inquiry explored how negative imagery influenced interpretation of ambiguous social situations. Examination of interpretative processes has particular relevance in social anxiety, as socially anxious individuals have been shown to exhibit a tendency to interpret ambiguous social events in a negative way (Clark and McManus, 2002). In Hirsch et al.(2003), participants were instructed to generate negative selfimagery while making "online" (i.e., as they read) interpretations of ambiguous social information. While participants assigned to a control (unrelated) task exhibited the usual non-threatening interpretation bias observed in non-anxious individuals (Hirsch and Mathews, 1997), those assigned to the negative self-imagery condition did not show such positive bias.

Interestingly, the link between imagery and bias was further shown to be reciprocal. In Hirsch et al. (2007), participants completed a repeated practice in accessing positive or negative interpretations of ambiguous social situations, a procedure known as "cognitive bias modification" (CBM; Mathews and Mackintosh, 2000). Following the completion of the CBM procedure participants were asked to imagine themselves in ambiguous social situations. Participants in the negative condition generated more negative self-imagery than those in the positive group, which demonstrated the reciprocal influence of bias on negative selfimagery. Additionally, when subsequently asked to imagine taking part in a stressful social situation, participants in the negative group rated their anticipated anxiety as being greater and their expected social performance as being poorer than those in the positive group. The bidirectional link shown between imagery and interpretation bias suggested the importance of examining the combined influence of cognitive biases in the maintenance of social phobia (Hirsch et al., 2006).

## **POSITIVE IMAGERY IN SOCIAL ANXIETY**

In line with the general emphasis in clinical research on negative cognitions and affect, the programme of research outlined above mostly focussed on delineating the influence of induced negative self-imagery on negative interpretation bias, ratings of social performance and social anxiety. The converse possibility that inducing positive imagery might lead to opposite and thus beneficial effects on social anxiety has been largely unexplored. There is accumulating evidence, however, that socially anxious individuals exhibit a range of positivity impairments, including reduced positive affect, absence of positive interpretation bias and deficits in approach-oriented behavior (Alden and Taylor, 2004; Kashdan, 2007; Weeks and Heimberg, 2012). Thus, targeting these positive deficits may represent an important target for future treatment of SAD.

Positive self-imagery represents a promising candidate to intervene on these positivity impairments in social anxiety. Mental imagery has been demonstrated to elicit stronger emotions than other forms of processing (Holmes and Mathews, 2010; Pictet and Holmes, 2013). This evocative power has further been shown to apply to both positive and negative emotions, leading to the suggestion that mental imagery may act as an "emotional amplifier" (Holmes et al., 2008a). Mental images have further been found to produce stronger emotions when they are generated from a field perspective, rather than from the perspective of an observer. This finding holds relevance in SAD given the tendency among socially anxious individuals to generate negative selfimagery from an observer perspective (Wells and Papageorgiou, 1999). Other properties of mental imagery with relevance to social anxiety have been identified. For example, imagining an event happening in the future has been shown to increase the perceived likelihood that the same event will actually occur in the future (Carroll, 1978). Further, generating positive imagery of the future has been shown to enhance motivation and goal-oriented behavior (Libby et al., 2007; Pictet et al., 2011). Taken together, these results suggest that promoting positive, field perspective imagery of the future in SAD may lead to improvements in anxiety symptoms, cognitive biases and motivation to engage in social behaviors.

Only a few studies to date have directly examined the impact of positive mental imagery in the context of social anxiety. In Stopa and Jenkins (2007) as well as Vassilopoulos (2005), participants who held a positive image in mind while giving a speech reported feeling less anxious and performed better than those who held a negative image. A limitation of this finding was that in the absence of a control condition, it was impossible to tell whether the effects found reflected the influence of positive imagery, negative imagery or a combination of the two. More recently, Hulme et al. (2012) explored the effects of experimentally inducing positive imagery or negative imagery on self-esteem, both during the imagery induction task and after performing a social threat task in which participants experienced social exclusion. Both high and low socially anxious participants who had held a positive image in mind reported higher levels of self-esteem at both times of the experiment than those who had held a negative image. A limitation of these findings was that the effects found were shortlived as confined to a single experimental session. Stopa et al. (2012) addressed this by testing whether an extended imagery practice involving seven daily sessions of positive imagery could lead to beneficial effects in socially anxious individuals. The imagery induction method used in the study was based on Hirsch et al.(2003) and consisted of the following two stages. Participants were first asked to generate a positive (or negative) image based on a social situation in which they had felt relaxed (or anxious). They were then asked to practice holding the positive (or negative) image in mind while listening to descriptions of ambiguous social situations (e.g., being called to reception in a Doctor's surgery). Results indicated that after 1 week's practice, participants in the positive condition reported higher levels of self-esteem and better performance ratings during a conversation with a stooge (in both subjective and objective assessments of performance).

The preliminary findings about positive imagery are encouraging but a replication on a clinical population is warranted. A common limitation in the research described was the absence of a control condition, which made the findings confusingly imputable to either negative or positive imagery, or a combination of the two. Further, little is known to date about the optimal way to promote positive imagery in SAD. The method employed in previous studies to induce positive imagery involved asking participants to recall a memory in which they had felt relaxed, then inviting them to close their eyes and ask them a series of questions designed to prompt imagery, such as how they thought they looked and sounded, how they felt and how they came across to other people. Whilst this method has been successful in producing the expected changes in previous research, there are many other possible ways to promote positive imagery and more research is needed to identify they key aspects of imagery than need to be harnessed. A potential caveat with regards to the methodology used was the questions used to prompt imagery (i.e., how they thought they look and came across to other people) may have favored the generation of observer perspective imagery. Encouraging the use of field perspective imagery (i.e., seeing the event through one's own eyes) is likely to produce stronger effects on negative and positive emotions, and counteract the natural tendency among socially anxious individuals to imagine themselves from an observer perspective. A second caveat with regards to the methodology used is that it may be too challenging for individuals with clinical levels of social anxiety to recall and elaborate on a time when they had experienced a positive social situation. This possibility is supported by recent evidence showing that social anxiety is associated with an impairment in generating detailed imagery of past positive events (Moscovitch et al., 2011). One possible way to overcome this could be to train the generation of positive imagery in response to hypothetical, future-oriented scenarios rather than to past memories. Imagining the future involves similar psychological processes to remembering the past (Schacter et al., 2007), with the additional benefit that it may help reduce anticipatory anxiety and boost motivation to engage in future social activities.

## **POSITIVE IMAGERY AND COGNITIVE BIAS MODIFICATION (CBM) IN SOCIAL ANXIETY**

The experimental research reviewed above evidenced a close and reciprocal relationship between imagery and negative interpretations of ambiguous social situations. In Hirsch et al. (2005), taking the perspective of a confident speaker rather than their own perspective was shown to block threatening inferences in people with high interview anxiety. However, it did not facilitate the generation of more benign interpretations. One possible explanation for this lack of effect on positive bias could be that participants may have struggled to adopt the perspective of a confident other as it was too far from what they would usually experience in similar situations. Another possible interpretation was that the induction procedure was too short-lived and thus not powerful enough to produce observable change in positive interpretation bias. By contrast, a more extensive practice of generating positive selfimagery in a CBM-I procedure could represent a more powerful way to produce changes in affect, cognition and behavior in a population of socially anxious individuals.

Only a few studies to date have explored the potential benefit of interpretation bias modification (CBM-I) for people with high levels of social anxiety (Murphy et al., 2007; Beard and Amir, 2008; Beard et al., 2011; Turner et al., 2011). Preliminary evidence supports the idea that CBM-I may be efficient at inducing a more benign (and less negative) interpretation bias and reducing anxiety symptoms in socially anxious individuals. However, very little is known about the durability of these effects as none of these studies included follow-up assessments. In the only attempt to combine CBM-I with mental imagery, Murphy et al. (2007) instructed high socially anxious participants to imagine themselves in a series of auditorily presented descriptions of social situations that started ambiguous but then were consistently resolved in a benign or negative way. Compared to a control condition where the same situations were presented but the outcome remained unspecified, participants who completed the benign interpretation training showed less negative and more positive interpretation biases, and further anticipated that they would be significantly less anxious in a future social situation. These effects were not imputable to group differences in state anxiety (as mood had been successfully equalized between the two groups following a filler task) and thus were directly due to the CBM-I procedure. Both the imagery and bias induction may have contributed to the positive effects found. At the time participants were presented with an upcoming social situation that could be potentially threatening (i.e., meeting two people they don't know for a 5 min conversation), those who had previously completed the positive interpretation training may have been influenced by their newly acquired bias and interpret the situation in a more benign way (congruently with the training). Alternatively, participants may have spontaneously generated a positive image of them taking part in the upcoming social situation and thus have anticipated less anxiety.

The present article defends the view that combining a repeated practice in generating positive imagery with a CBM-I intervention may be a helpful component in future treatment of SAD. A similar hypothesis has been suggested in the context of depression (Holmes et al., 2009). Depression and social phobia are highly comorbid disorders (Ohavon and Schatzberg, 2010) and share common positivity impairments, such as reduced positive affect, lack of positive interpretation bias and deficit in positive imagery (Rude et al., 2002; Holmes et al., 2008b; Werner-Seidler and Moulds, 2011).

The effects of imagery-focussed CBM on depression have been tested using analog and clinical samples of depressed participants (Blackwell and Holmes, 2010; Pictet et al., 2011; Lang et al., 2012). In imagery-focussed CBM, participants first completed an introductory session in which they were trained to use imagery in a specific way (i.e., vivid, from a field perspective) and then were given a practice session of the CBM tasks on the computer. Throughout the training, participants were repeatedly prompted to focus on imagining the scenarios with as much vividness as possible. Pictet et al. (2011) found that within an analog sample of dysphoric participants, those who had completed a positive imagery-focussed CBM exhibited greater improvements in positive mood and greater performance on a behavioral task assessing approach motivation and persistence than those who had completed a control imagery condition. In the first clinical test of imagery-focussed CBM, Blackwell and Holmes (2010) administered a multi-session program involving seven sessions of CBM to a small sample of participants with a current major depressive episode. Imagery-focussed CBM was found to lead to significant improvements in depressive symptoms and cognitive biases, and these improvements were maintained at 2 weeks follow-up. Although promising, the findings were limited by the absence of a comparison condition, which left open the possibility that "non-specific" factors as well as spontaneous recovery might have intervened.

Lang et al. (2012) addressed this limitation by exploring the impact of a multi session imagery-focussed CBM-I compared to a closely matched control version of the program in a sample of currently depressed patients. Participants in the positive condition showed significantly greater improvement in depressive symptoms and cognitive bias from pre- to post-treatment compared to those in the control condition, and these improvements were maintained at 2 weeks follow-up. Of relevance for a potential application of imagery-focussed CBM to people with social anxiety, participants assigned to the positive imagery condition in Lang et al. (2012) reported significant reductions in traitanxiety. More recently, Williams et al. (2013) tested the effects of an online version of imagery-focussed CBM in which seven daily sessions of CBM were followed by a 10-week internet-based cognitive-behavioral therapy (iCBT). Results showed that compared to participants assigned to a waitlist control, those assigned to the imagery-focussed CBM-I showed significant reductions in depressive symptoms, anxiety and distress. In addition, about a third of the participants showed clinically significant change after imagery-focussed CBM-I, while the proportion of clinical response reached 65% when combined with iCBT.

### **CONCLUSION**

SAD is a highly prevalent condition that causes considerable distress and significant functional impairments (Kessler et al., 2005). Effective psychological treatments exist but they are underutilized due to the lack of qualified therapists and to the reluctance of some socially anxious people to disclose personal information to a stranger. This had led to increasing calls for the development of easily accessible and effective treatments (Clark et al., 2009). CBM procedures represent promising candidates as their format allows great flexibility and accessibility, and requires minimal clinical involvement. Early findings from clinical studies (Beard et al., 2011; Brosan et al., 2011; Amir and Taylor, 2012) suggest that CBM-I could be beneficial for socially anxious individuals, but more research is needed to determine the most efficient way to deliver CBM-I in clinical samples. Further, future research should include follow-up assessments to evaluate whether the effects of CBM are maintained over time. The research reviewed in this article converges on the proposition that using mental imagery could enhance the efficacy of CBM-I in SAD. One way in which we "resolve" the ambiguity of a social situation is by imagining the outcome. Hence, increasing access to positive and vivid representations of social situations in a CBM-I program could be a promising target for future treatments of SAD.

## **REFERENCES**

Williams, A. D., Blackwell, S. E., Mackenzie, A., Holmes, E. A., and Andrews, G. (2013). Combining imagination and reason in the treatment of depression: a randomized controlled trial of internet-based cognitive bias modification and internet-CBT for depression *J. Consult. Clin. Psychol.* 81, 793–799. doi: 10. 1037/a0033247

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 July 2013; accepted: 20 January 2014; published online: 04 February 2014.*

*Citation: Pictet A (2014) Looking on the bright side in social anxiety: the potential benefit of promoting positive mental imagery. Front. Hum. Neurosci. 8:43. doi: 10.3389/fnhum.2014.00043*

*This article was submitted to the journal Frontiers in Human Neuroscience*.

*Copyright © 2014 Pictet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*


Meet the editors

Dr Vishwanath Gaitonde is currently appointed as a Senior Research Scientist at Cambrex High Point, NC, USA, where he designs, develops, and optimizes chemical processes for multi-kilo scale cGMP and nGMP production campaigns to support clinical trials in Phase I – Phase III. Prior to joining Cambrex, Dr Gaitonde was engaged in optimization of a lead molecule targeting broad spectrum viral respiratory disease in the Antiviral Me-

dicinal Chemistry Department, GlaxoSmithKline, NC, USA. Dr Gaitonde's PhD research was focused on the development of carbohydrate-based molecules as biochemical tools to probe the Mycobacterium tuberculosis GlgE mechanistic aspect. Additionally, his PhD research involved development of a novel amorphous polyester material based on sustainable chemistry.

Dr Partha Karmakar is currently appointed as a Research Scientist at the Washington University School of Medicine where his research involves development of a new therapeutic strategy for cancer treatment including development of small molecules and bioconjugates probes and an in-vitro and in-vivo efficacy study emphasizing clinical translation. Dr Karmakar was previously engaged in development and optimization of a lead molecule

targeting the bacterial colony growth mechanism in the Antibacterial Drug Discovery Department at GlaxoSmithKline – PA, USA. Dr Karmakar's PhD research involved development of glycopeptide-based vaccines and their in-vivo immunological evaluation.

Dr Ashit Trivedi is currently appointed as a Scientist in the Clinical Pharmacology Modeling and Simulations group at Amgen, USA, where his research work is extended into different therapeutic areas including oncology and neurosciences with a focus in cardiovascular diseases. As a clinical pharmacologist, Dr Trivedi participates in multifunctional study teams to design and conduct clinical pharmacology studies to support the global reg-

istration of pipeline drugs, perform PK and exposure-response analysis to inform dose selection, summarize and interpret PK, PK/PD, and clinical pharmacology results for internal/external presentations, study reports, and regulatory documents and interact with the line-management and different functions to implement strategies for drug label.

Contents

**Section 1**

**Section 2**

**Section 3**

*and Rossana C. Zepeda*

and Recent Applications

*and Grace Chitima Mugumbate*

*Iris Feria-Romero and Sandra Orozco-Suarez*

in Healthy Volunteers

*and Thales Kronenberger*

**Preface III**

Introduction **1**

**Chapter 1 3**

Computational Chemistry Advances in Modern Day Drug Discovery **11**

**Chapter 2 13**

**Chapter 3 27**

**Chapter 4 49**

Current View towards Pharmacokinetics in Drug Discovery **69**

**Chapter 5 71**

**Chapter 6 85**

Introductory Chapter: The Modern-Day Drug Discovery *by Partha Karmakar, Ashit Trivedi and Vishwanath Gaitonde*

Molecular Docking in Modern Drug Discovery: Principles

*by Aaftaab Sethi, Khusbhoo Joshi, K. Sasikala and Mallika Alvala*

Computational Deorphaning of *Mycobacterium tuberculosis* Targets *by Lorraine Yamurai Bishi, Sundeep Chaitanya Vedithi, Tom L. Blundell*

Revisiting Pharmacokinetics and Pharmacogenetics of Methadone

ADME Profiling in Drug Discovery and a New Path Paved on Silica *by Arne Krüger, Vinicius Gonçalves Maltarollo, Carsten Wrenger*

*by Natalia Guevara, Marianela Lorier, Marta Vázquez, Pietro Fagiolino,*

Past, Present, and Future of Molecular Docking *by Thuluz Meza Menchaca, Claudia Juárez-Portilla*

## Contents

Preface

Prevention and cure cases from various diseases reach a billion individuals every year. This is possible due to the organized efforts in the drug discovery program across the world led by pharmaceutical companies and academic institutions. Nevertheless, the majority of deaths globally are attributed to heart disease, stroke, pulmonary disease, lower respiratory infections, Alzheimer's, cancer, diabetes, and tuberculosis. One common aspect of these disease conditions is that they are preventable to some extent and a comparatively better quality of life can be provided to the patients with proper and timely treatment. To surmount this challenge, the drug discovery programs have advanced significantly in the past decade. This book, "Drug Discovery and Development – New Advances", brings together some of the recent progress in the field through the complex process, starting with computational modeling right through launching a drug in the market. The book aims to provide expert opinions and experiences from researchers encompassing diverse key topics critical in drug discovery and development. Case studies of some selected disease conditions will showcase the challenges and burdens faced by the pharmaceutical industries. The book covers the latest advances in molecular docking, pharmacokinetics, and new therapeutic options of some rare diseases along with pediatric drug discovery and development. Overall, the book represents a view

of the complex fabric of the modern-day drug discovery program.

**Dr. Vishwanath Gaitonde** Senior Research Scientist,

Cambrex High Point, Inc., High Point, NC, USA

> **Partha Karmakar** Research Scientist,

> > **Ashit Trivedi**

USA

USA

Department of Chemical Research and Development,

Clinical Pharmacology Modeling and Simulations Group at Amgen,

Washington University School of Medicine,


*4.3. Immunohistochemical Assay and qRT-PCR*

IHC staining was performed as described previously [23,24]. CSF antigens derived from the ND20, HY58, and GPE<sup>−</sup> strains were detected using DAB and the EnVisionTM peroxidase-conjugated polymer reagent (DAKO, Denmark). The results were scored according to the strength of straining as follows: +: 1–3 foci/section, ++: 4–10 foci/section, +++: >10 foci/section. The CSF RNA copy number within the organs of pigs was measured using quantitative real-time PCR (qRT-PCR) and calculated as log 10/g. The AnyQ CSFV qRT-PCR (Median Diagnostic Co. Cat No. NS-CSF-31, Korea) system and TaqMan probes targeting the 5'-UTR region with high specificity were used. The reaction conditions for qRT-PCR have been reported previously [24]. IHC staining was performed on the pigs showing most severe or mild clinical signs of each group. Whereas, qRT-PCR was performed on all pigs of each group.

**Author Contributions:** Conceptualization, S.C., V.P.L., and D.-J.A.; methodology, K.-S.K., J.-H.K., S.S., R.M.C., J.S., G.-N.P., and T.L.N.; writing/review and editing, B.-H.H., B.-K.P., and D.-J.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was supported by grants (Project Code no. I-1543083-2019-20-01) from the Animal and Plant Quarantine Agency and Ministry of Agriculture Food and Rural Affairs, Republic of Korea.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Ethical Approval and Consent to Participate:** All animal experiments were approved by Institutional Animal Cure and Use Committee (IACUC) of the Animal and Plant Quarantine Agency (APQA) in Ministry of Agriculture Food and Rural Affairs. All experiments were performed in secure biosafety level laboratories of APQA according to Chapter 1.1.4 biosafety and biosecurity guide in World Organization for Animal Health. 2018.


**5. Conclusion**

The extended abilities of laser ablation in liquid phase were presented through two topics. The first is nanoparticle formation of an organic material, which produced a colloidal solution of a small organic material. In a yellow pigment QQ, a systematic blueshift of the absorption peak corresponding to the decrease of particle size in colloidal solutions was discoverd. This dependence provides an easy estimation method of the averaged diameter of the ensemble that will be applied to organic devices by a wet process. Furthermore, the population of an excited triplet state through optical excitation might be one guideline to select and synthesize materials for laser fragmentation.

The second is hydrogen gas generation from solid carbon in water by a photochemical reaction. Even under a lower energy irradiation that achieves no plasma state, the irradiated site can be a reaction centre of energy conversion from light to a fuel gas, although the gas generation efficiency is very low. Simultaneously produced carbon-based nanoparticles possess a characteristic structure including those of diamond, nano-diamond, and diamond-like carbon, supplying new carbon materials from binchotan charcoal. A rise of temperature during the irradiated pulse duration was witnessed by the observation of white-light emission from the site ascribed to the blackbody radiation. Therefore, we conclude that the hydrogen generation induced by the laser pulse irradiation occurs under high-pressure and high-temperature conditions.

Laser ablation in the liquid phase is a useful technique to transform ordinary materials into functional ones under relatively mild condition. Although to date this technique has been applied to rather limited number of materials, further studies from various interests will extend the application field.