Joint rehabilitation goals are an important component for effective teamwork in the rehabilitation field. The activities and participation domain of the ICF provides a common language for professionals when setting these goals. Involving clients in the formulation of rehabilitation goals is gaining momentum as part of a person-centred approach to rehabilitation. However, this is particularly difficult when clients have an acquired communication disability. The expressive communication difficulties negatively affect the consensus building process. As a result, obtaining information regarding rehabilitation goals from professionals and their clients warrants further investigation for this particular population.This comparative study investigated clients and their assigned rehabilitation professionals' perception of the importance of ICF activities and participation domains for inclusion in their rehabilitation program. Twelve clients in an acute rehabilitation centre and twenty of their corresponding rehabilitation professionals participated in an activity using the Talking Mats™ visual framework for goal setting. Each participant rated the importance of the nine activities and participation domains of the ICF for inclusion in their current rehabilitation program.The ICF domains which consistently appear as very important across these groups are mobility, self-care and communication. Domains which consistently appear in the lower third of the rankings include spare time, learning and thinking and domestic life. Results indicate however that no statistical significant differences exist in terms of the individual domains across each of the participant groups. Within group differences however indicated that amongst the speech-language therapists and physiotherapists there was a statistical significant difference between spare time activities and communication and mobility.Findings indicate that consensus is possible amongst professionals and clients even within an acute-rehabilitation setting. In addition, the Talking Mats™ visual framework appears to be a valid protocol for including clients with acquired communication disabilities in the process of obtaining consensus during goal-setting. The concept of person-centred care is currently receiving attention in the rehabilitation literature . Person Establishing consensus is contingent upon establishing a common language which all members of the rehabilitation team (including the clients themselves) can use when describing health and health-related constructs . Person-In spite of this debate, component of the ICF have been successfully used to conceptualize meaningful participation in activities across numerous aetiologies and types of disability namely chronic disability , childreIn recent years, however, tools have been developed which attempt to translate elements of the ICF framework for use within rehabilitation settings which subscribe to the ethos of person-centred care, and client involvement in the process of goal-setting. Talking Mats™  is one sTo date, however, there is a paucity of published data that determines if consensus is possible between rehabilitation professionals and clients in an acute-rehabilitation centre. The acute stage of rehabilitation is a particularly important stage in the rehabilitation process as it is the first opportunity for rehabilitation professionals to develop authentic partnerships with these clients. Due to the length of time that many of these clients will be accessing rehabilitation services, it is crucial to employ person-centred care principles in the rehabilitation process in an attempt to ensure that client remains engaged in the rehabilitation process. Therefore in the present study clients and professionals at an acute-rehabilitation centre were asked to rate the importance of the nine ICF activities and participation domains in terms of their inclusion as a current rehabilitation goal, using the Talking Mats™ visual framework. This was viewed as an initial step in establishing rehabilitation goals which reflect the priorities of all team members.This study uses a comparative design . An adapEthical clearance was granted by the University of Pretoria and the research adhered to ethical guidelines stipulated by the University. Written consent was then obtained from the selected rehabilitation centre and all the candidates who met the selection criteria were invited to participate in the study. Participants were informed that their participation in the study was voluntary and that they may withdraw at any stage during the duration of the study without any negative consequences to their current rehabilitation program. Participants gave consent for the sessions to be recorded. These video recordings were used to establish procedural integrity.The rehabilitation centre which was the site for main data collection was purposefully selected and is funded by local government, has been in existence for longer than 5 years and serves individuals from a relatively large ethno-graphically diverse geographic region of the Western Cape Province of South Africa. Purposeful sampling was adopted in order to ensure that the site contained clients who had an acquired communication disability. All clients presented with an acquired communication disorder e.g. expressive aphasia, apraxia or dysarthria as a result of either traumatic brain injury (TBI) or stroke and were currently receiving rehabilitation services at the chosen rehabilitation centre. This ensured that the clients would provide relevant data which would assist with answering the aims of the research questions during the data collection procedures . All theTwelve clients and the 20 rehabilitation professionals treating them  participated in the study. The professionals described each client in terms of the clients' current motor, cognitive and communication skills using the Abilities Index . Figure All of the clients had some degree of motor involvement. The majority (n = 7) had mild expressive language disorder, but nevertheless passed the receptive language screening test. Three participants provided consent for the study, but did not pass the receptive language screening test and were therefore excluded from the study. Of the remaining clients five were male and seven were female. Seven of the clients were between 25-40 years of age, three were above 40 years of age and two were below 25 yrs of age. Seven of the clients were diagnosed as having had a stroke while five clients were diagnosed with a traumatic brain injury.The 20 professionals who participated consisted of six occupational therapists, seven physiotherapists, two speech-language therapists and five social workers. Half of professional participants (n = 10) had been employed in the field of rehabilitation for longer than five years and half (n = 10) had between one and five years of experience in the rehabilitation field. The majority (n = 15) of professionals, however, indicated that they felt very comfortable in discussing goal-setting priorities with clients. Nineteen of the 20 professionals were female.For the purpose of this research certain material was developed. The exemplars of the ICF activities and participation domains were developed  were asked to rate the three symbols they felt were the most representative symbols of each domain. The one group had both the graphic symbols and the words and the other group only had the symbols. The two lists were merged and the most frequently occurring symbols on both lists were developed into a composite list. Thereafter rehabilitation professionals n = 10) were asked to place the symbols on this composite list into the correct domains. The three items which were most consistently placed in the domain by most professionals were then selected to represent each domain during testing  so that individuals with communication difficulties may participate in the goal-setting process . Picturevery important, less important, and not important at all) were developed. A series of three trial items were developed, and included in the interview schedule in order to ensure that the participants understood the instructions and were able to complete the task that was required of them. The wording of the interview schedule for both groups of participants  was essentially the same, and covered exactly the same content. However, due to the fact that the professionals themselves did not display any motor, cognitive, or communication difficulties, they were not required to complete either the screening protocol or the trial items from the interview schedule.An interview schedule was compiled to ensure consistent presentation to each participant. The interview schedule provided a description of each of the 9 ICF activities and participation domains, as well as the question which asked the participant to rate the importance of each of the nine domains. A three-point Likert scale was constructed for use in the study, and pictorial representations for each of the options . During this test the researchers would also be able note what modifications would needed in order to accommodate the client's level of motor functioning during completion of the interview schedule.The aim of the pilot study was to determine the effectiveness of the screening materials and the interview schedule and . The material and procedural efficacy was also assessed and modified as needed. Five clients (three with a stroke and two with traumatic brain injury) as well as three occupational therapists and one physiotherapist participated. All the participants met the selection criteria proposed for the main study. Minor adaptations to the instructions and procedure of the interview were made following the pilot study.Once the rehabilitation clients gave their consent to take part in the study the screening tool was administered. If the participant successfully completed the screening and trial items, the interview was completed using the Talking Mats™ visual framework activity, according to the predetermined interview script. After completion of the Talking Mats™ the researchers checked that the client was satisfied with their choices and then captured a visual image of the completed mat with a digital camera. After all the clients completed the screening and Talking Mats™ activity, the same procedure was followed with the professionals working with these clients, excluding the administration of the screening protocol and trial items. The professionals completed a separate interview and corresponding Talking Mats™ for each of the clients to whom they provided rehabilitation services.All participants  were called into a room with minimal distractions. One of the researchers conducted the interview and was seated opposite the participant at the table. Another researcher operated the video camera and took a digital photograph of each participant's mat after it had been completed. This visual representation then formed the basis for the analysis of the data. Responses were converted into a raw score which were captured in an Excel spreadsheet. This was then analysed using relevant statistical procedures.The video recordings of thirty percent of the completed interviews were randomly selected and checked for procedural integrity by a B. Social Science (Hons) student, specializing in Psychology. An overall rating of ninety-one percent was obtained which indicates a high level of procedural integrity across the selected interviews.Once this had been established the mean and standard deviation  were comThe main aim of this study was to describe the importance ratings assigned to each of the ICF activities and participation domains by rehabilitation professionals and clients in an acute rehabilitation context utilizing the Talking Mats™ visual framework. The results will be discussed in terms of the rehabilitation priorities (those domains which were rated by a group of participants as being very important for inclusion in the current rehabilitation program). Similarities and differences between the participants groups will be explored. Importance ratings, or priorities, are described according to the mean score each group assigned to each of the nine ICF domains. Maximum scores that could be obtained for each ICF domain by each participant was 3 (indicating very important) and the minimum score was 1 . This section will conclude with a discussion regarding the relevance of using the Talking Mats™ visual framework for this population and within an acute care context.The mean scores for each of the nine different domains ranged from 1.82 (0.75) to 3.00 (0.00) and can be viewed in Table All participant groups  rated coping, communication, mobility, self-care, and relationships as important domains for inclusion in the rehabilitation program, as can be viewed in Table The results indicate a high degree of consensus in terms of those domains which participants' rated as important in the current rehabilitation program. The domains which are consistently rated as most important  appear to be primarily activity domains. Results from a study which used the ICF to identify priorities between professionals, parents and youth with cerebral palsy also indicated mobility and communication amongst the top priorities across all the participants . The domA Friedman analysis was then conducted; however no statistically significant differences were reported for the ratings across the participant groups. This supports the finding that priorities and non-priorities can be highlighted and agreed upon by all the participant groups who took part in this study. It is therefore feasible to deduce that consensus is indeed possible between clients and professionals within the acute-care setting. It also implies that professionals in the rehabilitation team are able to identify domains which were not primarily within their scope of practice as important domains (speech-language therapists identified mobility and self-care as important while physiotherapists identified communication as important).Each group of professional's responses were then compared across all of the clients that they treated to determine if any patterns exist in terms of the importance certain professional disciplines placed on certain domains. Results from this Friedman analysis indicated statistically significant differences between domain priorities exist in two professional participant groups, namely the speech-language therapists and the physiotherapists. Results can be viewed in Table For both of these groups the differences are between the spare time domain and the domains of communication and mobility. For the speech-language therapists this difference is also present between spare time and relationships. This trend seems to indicate that the domain of spare time is not considered as important to include as a rehabilitation priority in the acute-care setting by certain professionals. However if the activity and participation domains are indeed conceptually different as proposed by Whiteneck and Dijkers  and BadlThe use of the Talking Mats™ visual communication framework therefore appears to be an effective manner of communicating with people who have an acquired communication disorder in order to determine rehabilitation priorities within the acute stage of rehabilitation. This may possibly be due to the fact that the framework uses symbols to augment the verbal and written input traditionally given during activities such as goal-setting. The degree of overlap between professionals and clients regarding the important domains for inclusion indicate that this is a reliable method for assisting individuals with an acquired communication disability to participate in the identification of rehabilitation priorities. It appeared as if Talking Mats™ provided the clients the opportunity to express their views, which they would have had difficulty doing using a more traditional interview format. Murphy  reportedThis study has several strengths and limitations which will be discussed below. Due to the small number of participants  together with the fact that the main data collection occurred at a single site, means that the results must be generalized with caution. A further study with a larger number of participants would serve to further support the results obtained from this group of participants.Talking Mats™ is however, a well researched framework that enables people with acquired communication difficulties to actively participate in setting rehabilitation goals. This study provides initial evidence to suggest that the Talking Mats ™ visual communication framework is also appropriate for use within the acute-rehabilitation context. Results indicated that clients' priorities were in fact similar to those identified by the rehabilitation professionals, when using this framework. Professionals and clients are therefore, to a large extent, able to agree on the rehabilitation priorities within an acute-care setting. Four important domains were highlighted by both groups. These domains dealt primarily with activity limitations pertaining to mobility and communication. This information can be used to set rehabilitation goals which both the client and professional feel are meaningful. This is in keeping with a client-centred approach as it enables greater client self-determination and control, which enhances the potential for active and sustained participation within the rehabilitation program . AlthougIt is important to note that while Talking Mats ™ provides a simple framework to obtain clients' opinions; this methodology does possess a few constraints. Firstly, a considerable amount of planning is involved in the preparation of the material for use in the activity as indicated by Murphy . FurtherA final limitation of the current study is that there were a limited number of professionals who were involved in providing rehabilitation services to the clients. As a result the same professional provided rehabilitation to more than one client. This may have negatively impacted on the ability to focus on the priorities for a specific client, as professionals were required to complete an interview and Talking Mats ™ for each client they provided services to.Person-centred care focuses on the importance of cultivating the client as an active member of the rehabilitation team. When using the ICF's nine activities and participation domains as a common language it is possible to identify important domains to prioritize in the rehabilitation program. Findings from this study indicate that using a Talking Mats™ activity for individuals with an acquired communication disability, allows for these clients to participate in a meaningful way in the process of identifying important domains to be addressed during rehabilitation goal-setting. When comparing results between professionals and clients in an acute-care rehabilitation program, it is evident that a certain degree of agreement regarding both the domains of importance and non-importance is indeed possible. The majority of the domains which are viewed as important for inclusion at this stage of the rehabilitation process can be classified as activity domains . This would imply that the focus during the acute stage of rehabilitation is on addressing activity limitations as opposed to participation restrictions. When clients are able to reliably identify priorities and actively participate in establishing priorities for their own rehabilitation program it facilitates an increase in their sense of control and independence, which aligns with the tenets of a person-centred approach to rehabilitation.The authors declare that they have no competing interests.MH conceptualized the study, and participated in its design, statistical analysis and drafted the manuscript. MG and AVDM participated in its design, data collection and statistical analysis. All authors read and approved the final manuscript.Talking Mats™ symbols of the nine ICF domains and their exemplars.Click here for file


It has been more expensive per SSR locus recovered, however, particularly for genomes with few SSR loci, such as bird genomes. The longer but relatively more expensive 454 reads have been preferred over less expensive Illumina reads. Here, we use Illumina paired-end sequence data to identify potentially amplifiable SSR loci (PALs) from a snake , and directly compare these results to those from 454 data. We also compare the python results to results from Illumina sequencing of two bird genomes , which have considerably fewer SSRs than the python. We show that direct Illumina Seq-to-SSR can identify and characterize thousands of potentially amplifiable SSR loci for as little as $10 per sample – a fraction of the cost of 454 sequencing. Given that Illumina Seq-to-SSR is effective, inexpensive, and reliable even for species such as birds that have few SSR loci, it seems that there are now few situations for which prior hybridization is justifiable.Identification of microsatellites, or simple sequence repeats (SSRs), can be a time-consuming and costly investment requiring enrichment, cloning, and sequencing of candidate loci. Recently, however, high throughput sequencing (with or without prior enrichment for specific SSR loci) has been utilized to identify SSR loci. The direct “Seq-to-SSR” approach has an advantage over enrichment-based strategies in that it does not require  Constant advances in DNA sequencing technology and lower costs are driving innovation in the life sciences, and are having an especially large impact on the study of ecology, evolution, and population genetics. With these advances, traditional approaches to data generation and marker development require continual re-evaluation. For example, simple sequence repeats  have long been important in population genetic studies, but the identification of SSRs from non-model species previously required substantial and costly technical effort, and often returned far fewer loci than were required to address most population genetics questions adequately. This effort included creating libraries enriched for SSR loci, cloning, hybridization to detect positive clones, plasmid isolation, and Sanger sequencing. The application of next-generation sequencing approaches has recently made the cost of obtaining SSR loci less expensive and more efficient, allowing researchers to focus technical efforts on obtaining larger sample sizes appropriate to answer the population genetics questions being asked.Several research groups While the per-base cost of 454 sequencing has stayed relatively constant, the cost of obtaining Illumina sequence data has dropped substantially. Illumina sequences now can produce moderately long reads  and accommodate paired-end sequencing from both ends of ∼200–600 bp fragments. There have also been massive increases in the number of reads obtained per Illumina sequencing run. To take advantage of these advances, we implemented and tested a new approach, analogous to the previous 454-based method, utilizing Illumina paired-end sequencing to identify PALs (SSR loci and flanking PCR primer sites) without library enrichment or post-sequencing assembly of reads.Python molurus bivittatus), which has been shown to have a relatively high genomic frequency of SSR loci Centrocercus minimus, and Clark's Nutcracker, Nucifraga columbiana). We specifically chose to test the approach on birds because, among vertebrates, they have particularly low genomic SSR content We first applied this approach to detect SSR loci from the Burmese python  from a captive bred Burmese python was used as a source for genomic DNA . Total DNA was extracted using standard phenol-chloroform-isoamyl alcohol organic separation, precipitated with ethanol/sodium acetate, washed with 70% ethanol, and resuspended in TE buffer. A total of 10 ug of this DNA was used to make two 454 shotgun libraries, one with FLX shotgun adapters and a second with FLX-Titanium adapters, both prepared using the standard shotgun library preparation protocol and quality control steps (Roche). Data from these two libraries have been previously published An Illumina paired-end (IPE) shotgun library was also prepared from 5 ug of DNA extracted from the same python individual, using a previously published protocol In addition to the python, total genomic DNA was obtained from two bird species. For the Gunnison Sage-grouse, DNA was extracted from blood obtained from an individual trapped in Gunnison, Colorado . DNA was isolated using standard phenol-chloroform-isoamyl alcohol organic separation, precipitated with ethanol/sodium acetate, washed with 70% ethanol, and resuspended in TE buffer. For the Clark's Nutcracker, DNA was extracted from muscle tissue of an individual using the Wizard Genomic DNA Purification Kit (Promega). That individual Nutcracker had been trapped near Logan, Utah and used for behavioural experiments . Illumina paired-end libraries for both birds were prepared following the same protocol as for the python, and the resulting paired-end libraries had a similar mean size of approximately 325 bp.Sequencing of the python 454 libraries is described elsewhere PAL_FINDER_v0.02.03, to extract reads that contained perfect dinucleotide (2mer), trinucleotide (3mer), tetranucleotide (4mer), pentanucleotide (5mer), and hexanucleotide (6mer) tandem SSRs. Reads were identified as SSRs if they contained simple repeats of at least 12 bp in length for 2–4mers , and at least 3 repeats for 5mers or 6mers. The reads were then sorted by the monomer sequence of the repeat  and by the number of tandemly repeated units observed. Non-unique repeat motifs  and translated or shifted motifs ) were grouped together, so that there were a total of four unique 2mer repeats, 10 unique 3mer repeats, and so on. If multiple SSR loci were discovered in a single read, the locus was considered a compound repeat if the SSR had different motifs; they were considered a broken repeat if the SSR had the same motif. In relatively rare cases in which the same repeated motif occurred at the internal termini of both paired-end sequences, the microsatellite was considered to be a spanning read, and annotated as such.<1?tlsb=-.025w?>A Perl script was written, which we named PAL_FINDER_v0.02.03 is operated using a control file that determines parameter settings. The control file can be readily modified by the user to alter criteria for SSR identification. For example, the user can specify which type of reads (454 vs. Illumina) are to be analyzed, and if the program should attempt to design primers or simply count SSR loci. The user can also specify the minimum number of tandem repeats (for each n-mer size class) to be considered, and which n-mer size classes to search for (from 2mers to 6mers).The program PAL_FINDER_v0.02.03, which is freely available (see below).A common motivation for identifying new SSR loci is to use them for scoring allelic length variation. Thus, newly identified SSR loci are typically useful only if primers in the non-SSR flanking regions can be designed and used successfully for PCR amplification. We therefore screened reads with SSR loci for flanking regions with high-quality PCR priming sites. The primer-pair design process was automated to submit large batches of sequences to a local installation of the program Primer3 (version 2.0.0) PAL_FINDER_v0.02.03 program contains a large number of parameter settings that direct the primer design criteria of Primer 3. These include ranges and optimal values for primer length, melting temperature, and secondary structure. The user can readily modify these parameter settings in the PAL_FINDER control file.For the purpose of selecting primer sites, low complexity and simple repeat sequences were masked from sequences flanking SSR loci using the RepBase v14.01 database (the “simple.txt” library) PAL_FINDER uses the raw set of reads as a reference and counts the copy number of forward and reverse primers in this library. A further consideration is that while the forward and reverse primer sequences may have multiple copies in the genome, they can still produce a single distinct band for scoring SSRs if they occur close to one another only once or a few times. In other words, even if primer sequences are somewhat frequent in the genome sample, they may only rarely occur in close proximity (and thus produce a PCR product). To evaluate this, we counted how often each PAL primer pair co-occurred in a set of paired reads in our library of reads for each species. Thus, PALs can be further screened based on the copy number of primers and primer pairs, with the lowest frequencies indicating primers and pairs most likely to amplify a single locus. All these attributes of PAL primers are annotated for each locus in the output of PAL_FINDER. These attributes of primer copy number, together with their sequences, and the detailed detection of SSRs per locus are output in a combined tab-delimited. This allows the output of SSR loci with flanking primers to be sorted and filtered by a number of criteria that might interest researchers.A concern in identifying PALs is the copy number of the primer sequence in the genome. We addressed this by estimating the number of observed occurrences of identified primer sequences in the sequence set analyzed. Specifically, P. reticulatus) has been estimated to be 1.44 Gbp The genome size of the Burmese python is unknown, but a related python species , and we use these data to evaluate the performance of the 454 reads in the Seq-to-SSR approach. For direct comparisons with the 454 data, we subsampled the python Illumina data to include the same number of reads as the 454 data: 118,973 paired-end reads. Hereafter, we refer to these 454 and Illumina paired-end datasets as “454” and “IPE-119K”, as in For detailed comparisons of performance of 454 versus Illumina-based sequencing, we focused analyses on the python, for which we had both types of data from the same individual. In a previous study The 454 and IPE-119K python data contained the same number of reads and similar amounts of sequence , and were comparable in their ability to identify SSR loci and flanking primers . Just ovG-tests). Their basis is uncertain, but may be due to differences in sequencing performance in highly repetitive regions.To determine whether there were differences between the techniques based on SSR structure, we further examined SSR locus and PAL identification by repeat motif monomer size . More SSWe compared the effectiveness of Illumina Seq-to-SSR in two birds and the python using five million IPE reads each . As expected based on previous information on the relative abundance of SSR loci in bird and snake genomes e.g., 3mers, 4mers). The 4mers are most frequent in all three genomes, and particularly abundant in the python  have similar frequencies among the birds and the python, but many others differ substantially in frequency between the python and birds, and even between the two bird species  for filtering PALs, based on how their primer sequences (or their reverse complements) were repeated in the entire set of reads. The most stringent criterion was to take the product of the counts of the forward and reverse primers in all reads, where a product of “1” would mean that both the forward and the reverse primers were each only observed once in the entire read dataset. . The next stringency level was to take the minimum number of times either primer in a PAL occurred in the entire set of reads. With this filter set to “1”, for example, at least one of the two PCR primers chosen should be unique to the SSR locus targeted, and therefore lead to successful specific amplification. The least stringent criterion depended on the number of times that a pair of PAL primers was observed together, in the correct orientation, in paired reads. This is a direct estimate of how often they might occur in close enough proximity in the genome to produce amplifiable PCR products, but is the least stringent criterion because the amount of sequencing may be insufficient to detect repeated pairs. It is also possible that primer pairs might be amplifiable but are further apart than the lengths of the paired-end library, and are thus not detected.To decide what the cutoff numbers for each of these stringency criteria should be, we divided the numbers in each stringency criterion into classes based on 1, 2, or >2 observations. The proportions in each category for each stringency criterion were strikingly similar across the three species , with thPAL_FINDER_v0.02.03, and thus readily tuned. We then considered the same three stringency criteria as before  and longer repeat stretches . We refer to this highly selective set of loci as “Best PALs”. We note that while these particular criteria are somewhat arbitrary, these criteria are readily selectable using the control file of the program s before , each wis before . In birds before . Thus, ewww.EvolutionaryGenomics.com and www.snakegenomics.org). The Perl script (PAL_FINDER_v0.02.03) used to identify and analyze SSR loci is also freely available at http://sourceforge.net/projects/palfinder/and at the authors' web sites. An accessory script for preparing multiplexed IPE data for input into PAL_FINDER is available at the authors' websites. Identified sets of SSR loci, together with statistics for each locus, and primer sequences for PALs, are provided online as tab-delimited files for each of the three species analyzed, based on the full 5 million read datasets  is sufficient to identify tens of thousands of PALs, even in taxa such as birds that have low genomic SSR densities. Thus, with Illumina sequencing, there seems to be little justification for performing an intermediate step of hybridization (targeting specific SSR motifs) prior to sequencing Currently, the GAIIx is capable of producing ∼30 million reads per flow cell lane (1/8 of a flow cell), and the HiSeq is capable of producing ∼180 million reads per lane, with read lengths of up to 150 bp (GAIIx) and 100 bp (HiSeq) per read, for approximately $2500. In contrast, for a similar price the 454 platform would be expected to deliver approximately 300,000 reads (from a 1/4 70×75 mm picotiter plate), or 100× fewer than the GAIIx. Although the HiSeq platform offers 6-fold greater economy per paired read, the GAIIx platform offers longer read capability , and substantially more accurate base calling at lengths >50 bp. Given that ∼$500 of GAIIx sequencing of the python yielded over a half million identified SSR loci, and ∼175,000 PALs, it seems that the cost of the GAIIx is already sufficiently low that there is no great benefit to using HiSeq and its somewhat shorter and less accurate reads.As an example of the extreme economy of the method, the cost to sequence the IPE-119K dataset would have been ∼$10 on the GAIIx. Thus, for the python, 1/250 of a GAIIx lane yielded 4,129 PALs. Also, our shotgun library preparations utilized all independently purchased (“off the shelf”) reagents, rather than kits, and cost approximately $20 per sample. Considering that our results demonstrate similar performance for SSR locus and PAL identification on a read-by-read basis for 454 and IPE data, and a two to three order of magnitude difference in cost favouring IPE sequencing, IPE-based SSR identification by Seq-to-SSR is likely the preferred approach.The major benefit of the IPE Seq-to-SSR approach for evolution, population genetics, and linkage mapping studies is that it quickly, reliably, and inexpensively delivers an unbiased genome-wide characterization of SSR loci along with PALs and their primers. It also produces a rich dataset of randomly sampled sequences that can be used for other purposes, such as studying transposable element content, mitochondrial genomes, or other highly repeated DNA segments. Furthermore, unlike other methods, the Seq-to-SSR approach provides information on the possible repetitive nature of potential primers that no other approach provides. While other groups have used IPE sequencing to identify SSR loci from genomic libraries after enriching for SSRs Given the large number of loci identified, the Seq-to-SSR approach allows great flexibility to preferentially target loci with favourable characteristics. For example, longer SSRs PAL_FINDER, or are part of the output annotation for each locus and can thus be used to sort and filter sets of SSRs. To empirically demonstrate the effectiveness of the approach, we applied these same “Best PALs” filters to similar IPE data from a plant (Mimulus ringens), and empirically tested 48 highly stringent primer sets on four individuals. In the first attempt, 22 of these loci produced clearly distinguishable amplification products, and 21 were polymorphic. Another 9 were probably good polymorphic loci but require further PCR optimization. Thus, by sampling microsatellite loci on essentially a genome-scale, the IPE Seq-to-SSR approach provides excellent flexibility for researchers to choose microsatellite loci with a suite of favourable characteristics for their needs.Here, we provided one example of extremely stringent filtering of PALs to identify a set of “Best PALs” that have many of the above mentioned characteristics, as well as having longer repeat motifs (4–6mers). These strict criteria yielded hundreds of Best PALs in the birds and thousands in the python. These and many other features of loci are either adjustable parameters in our program Our approach and software for SSR loci primer identification should also be useful to rapidly characterize genomic SSR landscapes for comparative purposes. We used the earlier 454-specific version of this software to identify differences in the genomic SSR content among species of snakes. This led to the discovery that these differences were due to SSR-seeding by a particular family of transposable elements Dataset S1Potentially amplifiable loci output - Burmese python, Python molurus bivittatus (5 million read IPE dataset). This is a tab-delimited text file that can be readily imported into a spreadsheet.(TXT)Click here for additional data file.Dataset S2Potentially amplifiable loci output - Gunnison Sage-grouse, Centrocercus minimus (5 million read IPE dataset). This is a tab-delimited text file that can be readily imported into a spreadsheet.(TXT)Click here for additional data file.Dataset S3Potentially amplifiable loci output - Clark's Nutcracker, Nucifraga columbiana (5 million read IPE dataset). This is a tab-delimited text file that can be readily imported into a spreadsheet.(TXT)Click here for additional data file.Dataset S4Potentially amplifiable loci output - Burmese python, Python molurus bivittatus (119k IPE dataset). This is a tab-delimited text file that can be readily imported into a spreadsheet.(TXT)Click here for additional data file.Dataset S5Potentially amplifiable loci output - Burmese python, Python molurus bivittatus (454 dataset). This is a tab-delimited text file that can be readily imported into a spreadsheet.(TXT)Click here for additional data file.


This paper examines the impact of three components of an HIV prevention program  on consistent condom use by long distance truckers with paid and non-paid female partners in India. Data from 2,723 long distance truckers were analyzed using the propensity score matching approach. Based on utilization of services, the following categories of intervention exposure were derived: no exposure, exposure only to mid-media, exposure only to mid-media and interpersonal communication, exposure only to mid-media and project-run clinics, and exposure to all three intervention components. Compared to those who were not exposed to any intervention, exposure to mid-media alone increased consistent condom use with paid female partners by about ten percent. Exposure to mid-media and visits to project-run clinics increased consistent condom use with non-paid female partners by 26 %. These findings suggest that mid-media events and clinics were the most effective package of services to increase consistent condom use among the long distance truckers. Long distance truck drivers and their helpers (hereafter referred to as truckers) are considered to be at high risk for sexually transmitted infections (STI) and HIV in many parts of the world, including Asia –4, AfricIn India, recent evidence shows that around one-fourth of truckers have sex with female sex workers (FSWs)  and lessDue to high risk behaviours, high STI and HIV prevalence, and ability to transmit infections to new geographic areas, HIV prevention interventions among truckers started in early 1990s with aim to raise the awareness through group discussion on matters related to HIV, using posters and pamphlets with positive AIDS messages, street theatres and puppetry, and distribution of condoms . Some inThe Kavach intervention was initiated in 2004 in 36 transshipment locations across India covering the major national highways along six routes, namely, North–East, North–West, North–South, West–South, South–East and West–East. Transshipment locations are places where transporters and brokers operate to link truckers with clients who need to transport goods, and routes are road corridors on which truckers travel.The Kavach intervention was redesigned in 2006 by halving the number of implementation sites from 36 to 17 to focus on the major truck halt points in nine Indian states. Unlike the other HIV prevention programs to cover large number of intervention sites for truckers coverage, the redesigned program aimed to provide maximum coverage to truckers by working with high intensity program  at major transshipment locations in the country where large number of truckers halt and wait for their consignments.The Kavach intervention had three main components: mid-media events; peer-led dialogue based interpersonal communication (IPC) and project-run clinics. Mid-media events included street plays, health games, film shows, truckers’ festivals and the distribution of audio cassettes/compact discs. Mid-media events aimed to communicate messages on safe sex practices to truckers and motivate them to visit project-run clinics. Trained professionals conducted about ten street plays every month at each intervention site. Health games were conducted twice a day and film shows were organized once a week at each intervention site. Truckers’ festivals were large-scale events conducted annually to create an interest among brokers, transporters and truckers in the program. Audio cassettes/compact discs with catchy local songs interspersed with spoofs on popular film actors delivering HIV prevention messages and endorsing services at project-run clinics were distributed to truckers so as to reinforce positive messages on HIV when they travel on the highway. The project monitoring data indicate that nationally around 150,000 audio cassettes/compact discs were distributed every year.In the Kavach intervention, IPC included one-to-group discussions by trained peer educators who were either active truckers or ex-truckers. About 10–12 IPC sessions were conducted every day at each intervention site. In order to reinforce messages already received in the mid-media events, those messages were repeated during the IPC sessions.One static clinic and two mobile clinics were established at each intervention site to provide services at the door-step of truckers’ halting points. To avoid becoming stigmatized as STI clinics, the project clinics treated a range of general health ailments in addition to providing counseling services by a qualified counselor on safe sex practices, correct and consistent condom use, and diagnosis and treatment of STI so that truckers would not hesitate to visit these clinics. Following the McDonald’s business franchisee model, where the look and service of outlets do not differ across any location , projectOf the 17 intervention sites, six were purposively chosen as survey sites for data collection. The selected sites were located at Ghaziabad and Delhi in northern India, Indore in central India, Mumbai and Pune in western India, and Bengaluru in southern India. These sites were chosen because truckers from different parts of the country wait at these places for a considerable length of time for new consignments. Data were collected during February–August 2009 in two phases at each selected site in order to cover wider group of truckers’ population. A total sample size of 2,800 truckers was estimated for the survey.The total sample size was distributed across the six transshipment locations chosen for the survey in proportion to the number of truckers available at those transshipment locations. Parking areas for trucks were considered as clusters in each transshipment location for the multistage cluster sampling and all clusters were selected for the survey. The registration number of the trucks parked in each cluster on the day of the survey was listed and a systematic random sampling approach was used to select the trucks. The main driver or helper of the selected trucks was approached to participate in the survey.Respondents were selected only if they met the following three criteria: (i) aged 18 years or more; (2) had worked as a trucker for the past 2 years or more; and (3) were working in a truck with a national permit . Truckers who had already participated in the survey (either in an earlier phase of the survey or in the same phase) were not interviewed again.A pre-coded questionnaire translated into the local language was used to collect data by trained native speakers. A total of 2,810 truckers were interviewed in the survey. Of these, cases who were traveling on the South–East route ; cases eA comprehensive informed consent process was followed. Respondents were informed about the study including the duration of the interview (approx. 30 min), and their queries addressed before written consent was taken. To protect confidentiality all questionnaires were entirely anonymous and the names and addresses of respondents were not recorded. To maintain privacy, interviews were either conducted inside the truck or in a secluded public area such a corner road where others would not be able to listen to the interview. Participants were not given any monetary compensation for their time in the study.The socio-demographic characteristics considered in this paper were age (completed years), formal schooling , marital status , usual place of residence , route generally travelled , duration of work as a trucker (completed years), occupation  and availability of music player in the truck . Formal schooling was defined as the ability to both read and write.Exposure to mid-media was defined as exposure to at least one mid-media events  in the past 12 months . Exposure to IPC was defined as participation in at least one IPC session in the past 12 months . Exposure to project-run clinics was defined as at least one visit to a project clinic (either static or mobile) in the past 12 months . Exposure to any intervention was defined as exposure to at least one of the three intervention components, namely, mid-media, IPC and project-run clinics in past 12 months.To measure exposure to different intervention components in the past 12 months, a variable with the following five mutually exclusive categories were derived: no exposure, exposure only to mid-media, exposure only to mid-media and IPC, exposure only to mid-media and project-run clinics, and exposure to all three intervention components.Consistent condom use with paid female partners and non-paid female partners was the primary outcome variable measuring HIV-related risk behaviour. A paid female partner was defined as a woman to whom the respondent had paid cash in exchange for sex. A non-paid female partner was defined as a woman to whom the respondent was not married and did not pay cash in exchange for sex. Consistent condom use with a sexual partner was defined as condom use in every sexual encounter with that particular partner .2 test.Basic descriptive statistics  were presented to describe participants’ socio-demographic characteristics, exposure to the intervention, and outcome indicators. The bivariate association between the categories of intervention exposure and outcome indicators was examined using χThe propensity score matching (PSM) approach was adopted to examine the impact of truckers’ exposure to any intervention as well as exposure to different categories of intervention on consistent condom use by them with paid and non-paid female partners. Studies indicate that when only observational data are available without a valid control group, the PSM approach can be adopted to evaluate the impact of intervention exposure on an outcome –34. The The observed socio-demographic characteristics including age, literacy, marital status, occupation, duration of work as trucker, time away from family, and route category were considered as the covariates because these characteristics have been found to correlate with truckers’ sexual risk behaviors in India , 35, 36.Matching was carried out using the radius method in which each exposed trucker was matched only with the unexposed truckers whose propensity score fell into a predefined neighborhood of the estimated propensity score of the exposed trucker . The pre2 test (for percentages) and unpaired t test . The overall covariate imbalance of the model was examined by testing the joint significance of all the regressors (ability of covariates to predict exposure to any intervention) using the likelihood ratio test before and after matching.The key assumption in the PSM approach is that conditional on the propensity score, assignments to exposed and unexposed groups can be considered as random . One tesThe PSM approach enabled us to estimate the average effect of intervention exposure among exposed truckers (difference in condom use among exposed truckers and matched unexposed truckers) and the average effect of intervention exposure among unexposed truckers (difference in condom use of unexposed truckers and matched exposed truckers). The average effect of intervention exposure among exposed truckers measured the impact of intervention on exposed truckers whereas the average effect of intervention exposure among unexposed truckers measured the impact that the interventions would have had on unexposed truckers if they were exposed. These two effects were weighted by the proportion of truckers exposed and unexposed respectively to arrive at the impact of intervention exposure on consistent condom use which measured the increase in consistent condom use due to intervention exposure , 32. As Following these steps, we first examined the impact of exposure to any intervention on condom use by comparing the consistent condom use among truckers exposed to any intervention with that among matched unexposed truckers. Since exposure to mid-media was common among all the exposed truckers, categories with significant impact on outcomes were examined against exposure only to mid-media for assessing the additional impact different components. Analyses were conducted using the statistical software STATA (version 11).Mean age of the respondents was about 30 years. Of the 2,723 respondents, more than three-fourth (83.4 %) had formal schooling, nearly two-third (66.6 %) were currently married, and about one-fifth 22.1 %) were working as helpers. A total of 991 (36.4 %) truckers were not exposed to the intervention in the past 12 months, 651 (23.9 %) were exposed only to mid-media, 188 (6.9 %) were exposed only to mid-media and IPC, 449 (16.5 %) were exposed only to mid-media and clinics, and 444 (16.3 %) were exposed to all three intervention components. About 45 % of respondents reported sex with paid female partners in the past 12 months and almost three-fourths (74.1 %) reported using condoms consistently in such sexual encounters. About 22 % of reported having sex with non-paid female partners and less than half (41.8 %) had used condom consistently in such sexual acts  . Consistent condom use with non-paid female partners also varied from about one-third (31.8 %) among truckers exposed only to mid-media and IPC, to almost half among those exposed to mid-media and project-run clinics (54.9 %) and those exposed to all three intervention components (50.9 %) .Consistent condom use with both paid and non-paid female partners was significantly higher among truckers exposed to any intervention than those with no intervention exposure  as well as among those reporting sex with non-paid female partners . Similar results were obtained while comparing socio-demographic characteristics of all unexposed and matched unexposed truckers with the socio-demographic characteristics of truckers exposed to different categories of intervention exposure (results not shown in tabular form but available upon request).Among truckers who had sex with paid female partners, those exposed to any intervention differed significantly from all unexposed truckers in terms of almost all the socio-demographic characteristics Table . Howeverp = 0.001) and consistent condom use with non-paid female partners by 12.4 % . Exposure to mid-media alone increased consistent condom use with paid female partners by 9.9 %  as compared with no exposure; however, this was not found to have an impact on consistent condom use with non-paid female partners. Similarly, exposure to only mid-media and project-run clinics increased consistent condom use with paid female partners by 11.4 %  and consistent condom use with non-paid female partners by 26.0 %  when compared with no exposure.Table p < 0.001) as compared to exposure to mid-media alone. Similar results about the additional combined impact of IPC and project-run clinics were found while comparing exposure to all three components with exposure to mid-media alone.The additional impact of project-run clinics on consistent condom use with paid female partners was not evident when compared with exposure to only mid-media. However, visits to project-run clinics increased consistent condom use with non-paid female partners by about 20 %  (Table p = 0.044). Similar results were obtained for estimated impact of exposure to different categories of intervention exposure on consistent condom use with paid and non-paid female partners.The estimated impact of truckers’ exposure to any intervention on consistent condom use with paid female partners was insensitive to a bias introduced by unobserved confounders that increase the odds of exposure to any intervention up to 30 %  to motivate truckers to use condoms with non-regular female sexual partners.The percentage of truckers exposed to the intervention as observed in this study was found to be higher than that found in a large-scale survey among long distance truck drivers conducted during 2009–10 in India . This maOn the other hand, the levels of consistent condom use with paid female partners (74.1 %) and non-paid female partners (41.8 %) as found in this study are consistent with other large-scale study among long distance truckers in India which found that about 74 % of the truck drivers used condoms consistently with paid female partners whereas about 37 % of them used condom consistently with non-paid female partners . In receThis study has some limitations, which should be considered when interpreting the results. First, findings are based on self- reports, which are vulnerable to social desirability and recall biases. The survey measured a limited number of socio-demographic characteristics and did not include some of the important variables such as income and alcohol consumption, which may confound the results. Finally, although the PSM approach was used to establish a causal relationship between exposure to intervention and consistent condom use, this should not be considered an alternative approach to randomized trials with valid case and control groups.In conclusion, the study documents that the Kavach intervention resulted in a significant increase in consistent condom use with both paid and non-paid female partners. Exposure to mid-media alone substantially increased consistent condom use by truckers with paid female partners whereas exposure to project-run clinics had a significant impact on increasing condom use with non-paid female partners. These findings suggest the need for intensive mid-media events to increase awareness of HIV and easily accessible clinics that provide counseling on safe sex practices and STI treatment services to truckers to increase the adoption of consistent condom use with non-regular female partners. It also highlights the need to understand truckers’ risk perceptions, needs and the barriers to the uptake of project-run clinic services even after repeated attempts to promote behavior change.


Magnolia fargesii and is known to inhibit AR activity. To assess scopoletin's ability to mitigate sugar cataract formation in vivo, we studied its effects in a rat model of dietary galactose-induced sugar cataracts. Galactose-fed rats were orally dosed with scopoletin (10 or 50 mg/kg body weight) once a day for 2 weeks. Administering scopoletin delayed the progression of the cataracts that were induced by dietary galactose. Scopoletin also prevented galactose-induced changes in lens morphology, such as lens fiber swelling and membrane rupture. Scopoletin's protective effect against sugar cataracts was mediated by inhibiting both AR activity and oxidative stress. These results suggest that scopoletin is a useful treatment for sugar cataracts.Cataracts are a major cause of human blindness. Aldose reductase (AR) is an important rate-limiting enzyme that contributes to cataract induction in diabetic patients. Scopoletin is the main bioactive constituent of flower buds from  Cataracts are the leading cause of blindness worldwide. Hyperglycemia and diabetes increase the risk of developing cataracts . CataracAlthough cataracts can be successfully treated with surgery, it remains important to find nonsurgical treatments for this condition. Synthetic AR inhibitors (ARIs) have been studied to treat diabetic cataracts. The use of traditional medicines, which are mainly derived from plant sources, has remained critical for managing many chronic diseases . ConsumiMagnolia fargesii; this plant has been used to treat various inflammatory diseases as a component of traditional Chinese medicines ) method .Lens sections were deparaffinized and hydrated by sequential immersions in xylene and graded alcohol solutions. The slides were placed in 10 mM sodium citrate buffer (pH 6.0) and autoclaved at 121°C for 10 min. Sections were then blocked with normal serum obtained from the same species with a secondary antibody developed to block nonspecific staining. The sections were first labeled with mouse anti-AR antibody  overnight at 4°C. After washing, the sections were labeled with fluorescein-isothiocyanate-(FITC-) conjugated goat anti-mouse IgG  for 1 h at room temperature. Finally, the slides were analyzed using fluorescence microscopy . The negative controls for immunostaining were run by incubating the sections with nonimmune serum instead of the primary antibody.The results were statistically evaluated using a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test using GraphPad Prism 5.0 software .P < 0.01). Therefore, scopoletin slowed the development of galactose-induced cataracts.During the cataract analysis, all of the animals fed on the galactose diet developed mature cataracts after two weeks  . Polyolsin vitro [ARIs, such as sorbinil, prevented sugar cataractogenesis in experimental animals , 26. Amoin vitro . Recentlin vitro , 29. Couin vitro . Based o50 value of 4.32 μg/mL [μg/mL and reached approximately 40 minutes after administering 50, 100, and 250 mg/kg scopoletin in rabbits, respectively [μg/mL after administering 50 mg/kg scopoletin in rats [in vitro results, we chose 10 and 50 mg/kg doses of scopoletin to evaluate its anti-AR activity in rats. We found that the effective dose of scopoletin was 50 mg/kd, agreeing with the in vitro result.In our previous study, scopoletin had an excellent inhibitory activity on AR, displaying an IC32 μg/mL . Liu et ectively . The pea in rats . Based oThe enzymatic distribution of AR activity was supported by AR's localized immunofluorescence. In human and rat lenses, AR is primarily localized in the epithelial and superficial cortical fiber cells . In galaAlthough the prevention of sugar-induced cataractogenesis by ARIs appears to be caused by AR inhibition, the osmotic hypothesis might not fully explain diabetic cataracts in human subjects because, even during severe hyperglycemia, the examined tissues, including the lens, did not have sorbitol levels >2 mM . AntioxiIn summary, this study reveals that scopoletin may exert beneficial/protective effects during the sugar cataract development. Scopoletin inhibits the AR activity, polyol accumulation, and reduction of the GSH levels. We suggest that the scopoletin may be particularly useful in treating sugar cataracts.


Recent evidence suggests that transient hyperglycemia in extremely low birth weight infants is strongly associated with the occurrence of retinopathy of prematurity (ROP). We propose a new model of Neonatal Hyperglycemia-induced Retinopathy (NHIR) that mimics many aspects of retinopathy of prematurity. Hyperglycemia was induced in newborn rat pups by injection of streptozocine (STZ) at post natal day one (P1). At various time points, animals were assessed for vascular abnormalities, neuronal cell death and accumulation and activation of microglial cells. We here report that streptozotocin induced a rapid and sustained increase of glycemia from P2/3 to P6 without affecting rat pups gain weight or necessitating insulin treatment. Retinal vascular area was significantly reduced in P6 hyperglycemic animals compared to control animals. Hyperglycemia was associated with (i) CCL2 chemokine induction at P6, (ii) a significant recruitment of inflammatory macrophages and an increase in total number of Iba+ macrophages/microglia cells in the inner nuclear layer (INL), and (iii) excessive apoptosis in the INL. NHIR thereby reproduces several aspects of ischemic retinopathies, including ROP and diabetic retinopathies, and might be a useful model to decipher hyperglycemia-induced cellular and molecular mechanisms in the small rodent. Retinopathy of prematurity (ROP), is one of the major causes of visual impairment in children. It is characterized by an initial retardation of the progression of vascular growth toward the retinal periphery which predisposes to abnormal compensatory neovascularisation In diabetic adults, adverse effects of poor glycemic control and hyperglycemic state on retinal vasculature and function are well-known The objectives of this work were to characterize the effect of a moderate, clinically relevant, hyperglycemic phase on vascular development. Our results show that neonatal hyperglycemia very reproducibly induces severe inflammation, inhibition of physiological angiogenesis and neuronal degeneration in a very short period of time. Neonatal hyperglycemia retinopathy (NHIR) might be useful to help decipher hyperglycemia-induced cellular and molecular mechanisms in the retina that lead to diseases such as ROP and DR.All experimental protocols and procedures were approved by the local animal care ethics committee “Comité d'éthique en expérimentation animale Charles Darwin” (N° p3/2008/54) and met the INSERM guidelines.One day-old Lewis rat pups were used for the experiments. Pregnant animals were purchased from Janvier - Le Genest- St-Isle, France. Animals were housed at local animal facilities under 12/12 hours light/dark cycles and fed ad libitum. The number of pups was culled to 8–10 per litter at P1 by random selection.The model of neonatal hyperglycemia was adapted from previously published studies Pups were sacrificed serially by decapitation at P3, P4, P5, P6 and P7 (2 to 6 days after the STZ injection). Some rat pups were also assessed at P21, when the maturation of the retina is complete and the hyperglycemia has resolved. After sacrifice, eyes were removed and retinas were dissected and fixed in 4% paraformaldehyde for 20 minutes at room temperature prior to flatmounting or OCT (Tissue Tek®) embedding. Intraretinal vascularization and microglial cell density were assessed on whole retinal flatmounts. Retinal capillaries were labeled according to previously described standard immunohistochemical procedures OCT-embedded retinas were cryosectioned and sections through the optic nerve were used for immunolabeling. Frozen sections were stained according to previously described standard procedures. In addition to the antibodies used on retinal flatmounts, the following primary antibodies were used to study retinal cells type on cryosections: anti-Ap2α antibody  was purchased to Developmental Studies Hybridoma Bank ; mouse anti-glutamine synthetase antibody  was obtained from Millipore , and rabbit anti-protein kinase Cα antibody (PKCα)  from Santa Cruz . Anti-calretinin antibody  was used to label horizontal cells, and anti-Rho4D2 (kind gift of Dr D. Hicks) to label rods. GFAP antibody  was used to label astrocytes. The corresponding Alexa–conjugated secondary antibodies  were used to reveal the primary antibodies. The FITC conjugated-peanut agglutinin (PNA) was used to label cone photoreceptor . Sections were counterstained with DAPI.2) using the “angiogenesis tube formation” MetaMorph add-in . Parameters were set to select vessels between 3 and 40 µm. Vascularized areas were measured and expressed as percent of total retinal area Sections and flatmount images were captured with a DM5500 microscope  equipped with an ORCA ER Hamamatsu camera and analyzed by MetaMorph software . For each retina, tube length and branching of capillaries, and vascular density were measured on a minimum of 6 representative images , and retinas were immediately dissected and fixed in 0.5% glutaraldehyde, 4% paraformaldehyde PBS for 2 hours, dehydrated, and mounted in HistoResin. Sagittal, oriented sections (5 µm), crossing the inferior pole, optic nerve, and superior pole were cut , and stained with toluidin blue. Sections were photographed with a light microscope  and numbers of rows of the outer nuclear layer as well as number of the nuclei of the inner nuclear and ganglion cell layers were quantified at different distances from the optic nerve to the periphery.Pups exposed to hyperglycemia and their normoglycemic controls were sacrificed at P6. Retinas were dissected and total RNA was extracted using the Nucleospin RNAII Kit . Retrotranscription was performed using superscriptII . Real-time PCR was performed using 7300 Real-Time PCR System  in a 20 µl final volume with Power SYBR Green PCR Master Mix  and 0.25 µM primers. All samples were run in triplicate. Primers used for real-time PCR analysis are available upon request.p<0.05 was considered as statistically significant.All data are reported as mean +/− SEM unless otherwise indicated. Data were analyzed by unpaired t-test or one- and two-way ANOVA with Bonferroni post-tests, according to the number of groups compared . Hyperglycemia was induced by low STZ dose intraperitoneal injection at ages during which both the neural retina and the retinal vasculature are immature 2 . At P21, vessel density of the superficial plexus was evaluated. No differences were observed between control and STZ retinas . Activated MC/MPs express inflammatory cytokines such as CCL2, TNFα, and IL-1β in experimental diabetes Ccl2 (2.54+/−0.45 vs 0.92+/−0.21), Tnfα (2.47+/−0.47 vs 1.00+/−0.27), and Il-1β (3.35+/−0.68 vs 1.00+/−0.60) in the STZ-injected hyperglycemic group compared to controls  (iNos was undetectable in both groups (data not shown).Microglial cells (MC)/macrophages (MP) have been shown to be activated in experimental and human diabetic retinopathy  control  and hype control . Iba1+ coundness , a shortoundness . None oft-tests) . iNos waHere we show that clinically relevant hyperglycemia during retinal development significantly inhibits retinal angiogenesis, induces neuronal cell death and leads to MC/MP infiltration and inflammatory cytokine production. Neonatal Hyperglycemia-induced retinopathy is an inexpensive, reproducible and quantifiable model that mimics retinopathy of prematurity.Our goal was to develop a model of clinically relevant neonatal pathological hyperglycemia, with sustained levels of hyperglycemia at the time when angiogenesis and neuronal development take place in the retina We induced a moderate diabetic state with low mortality and little impact on body weight gain , which iSTZ is a widely accepted tool for examining the mechanisms of diabetic retinal injury and potential therapeutic interventions as shown by its extensive use in adult animals Elevated blood glucose concentration in the neonatal period has recently been recognized as an independent risk factor for development of ROP in very low birth weight infants ROP leads to long-term impairment of retinal function Ccl2, Tnfα and Il-1β overexpression reflects the pro-inflammatory environment induced by the hyperglycemia. In NHIR inflammation concurs with inhibited vascularization and neuronal cell loss similar to ischemic DR and ROP. If and how microglial cell activation, macrophage recruitment and proinflammatory cytokine release influence angiogenesis and neuronal apoptosis in NHIR is currently under investigation.Recent data suggest that inflammatory processes may participate in the pathophysiology of ROP, since inflammatory cytokines have been found to be increased in ROP eyes We believe that NHIR provides a new model to study inflammatory cascades during retinal development and will help elucidate mechanisms of inflammation-induced damage to the eyes of premature infants. Diabetic patients, and adult rodent diabetic model, also show early retinal microglial cell activation Our model reproduces aspects of ROP (inhibited angiogenesis) and common aspects of DR and ROP . In contrast to the OIR model, which displays little inflammation, inflammation in NHIR is a prominent feature and might help decipher the inflammatory aspect of hyperglycemia-induced neuronal and vessel abnormalities in ROP. Compared to other models of rodent diabetic retinopathy, our model has the advantage of being short, having a pronounced phenotype, and not requiring insulin treatment for animal survival.


Giardia duodenalis (g14-3-3), are consistent with this hypothesis, but whether g14-3-3 is functionally homologous to the epsilon isoforms is unknown. We use inter-kingdom reciprocal functional complementation and biochemical methods to determine whether g14-3-3 is structurally and functionally homologous with members of the two 14-3-3 conservation groups of the metazoan Drosophila melanogaster. Our results indicate that although g14-3-3 is structurally homologous to D14-3-3ε, functionally it diverges presenting characteristics of other 14-3-3s. Given the basal position of Giardia in eukaryotic evolution, this finding is consistent with the hypothesis that 14-3-3ε isoforms are ancestral to other family members.The 14-3-3s are small acidic cytosolic proteins that interact with multiple clients and participate in essential cellular functions in all eukaryotes. Available structural and functional information about 14-3-3s is largely derived from higher eukaryotes, which contain multiple members of this protein family suggesting functional specialization. The exceptional sequence conservation among 14-3-3 family members from diverse species suggests a common ancestor for 14-3-3s, proposed to have been similar to modern 14-3-3ε isoforms. Structural features of the sole family member from the protozoan  The 14-3-3s comprise a family of small acidic polypeptides, which form dimers and are ubiquitous in all eukaryotic kingdoms Giardia duonenalis , a worldwide parasite of the upper part of the small intestine of mammals including human Drosophila melanogaster 14-3-3s, asking for interspecies functional complementarity.Another remarkable characteristic of 14-3-3s is the high amino-acid sequence similarity among orthologs from different species, rather than with other isoforms of the same organism Giardia belongs to the diplomonads, a group of anaerobic/microaerophilic binucleated flagellates. The highly divergent Giardia spp. are considered candidate early-branching eukaryotes and provide unique opportunities for gaining insights into key events of eukaryotic evolution D. melanogaster has only two 14-3-3-encoding genes leonardo results in three spatially restricted and apparently functionally distinct isoforms The protist genus In addition to alternative splicing, posttranslational modifications are utilized to increase the number of distinct isoforms temporally or cell-type-specifically, to cope with functional diversity when limited by the number of distinct 14-3-3 encoding genes in a genome Drosophila and human 14-3-3ε isoforms, we investigated whether it is functionally orthologous to D14-3-3ε and conversely whether any of the fly 14-3-3 proteins function in G. duodenalis (henceforth referred to as Giardia). The two systems are ideally suited for this approach as they are highly distant evolutionarily, have well developed genetic arsenals and flies have only two 14-3-3 genes to the single protozoan counterpart. Of the three fly 14-3-3ζ (LEONARDO) isoforms, we chose LEOII because similar to D14-3-3ε it is broadly expressed spatially and temporally GiardiaBecause of g14-3-3s similarity to G. duodenalis strain WB-C6 were axenically grown and encystation induced as previously described Giardia lines were generated by electroporation, selected and maintained under 100 µM puromycin .Trophozoites of Drosophila melanogaster were cultured as described 6xHis-g14-3-3 on chromosome 3 were recombined onto this chromosome carrying the ex4D14-3-3 mutant allele Giardia-expression vectors, the FLAG epitope sequence was introduced by PCR at the 5′ end of D14-3-3ε and D14-3-3ζ(leoII) coding sequences using the primers: D14εForw:5′CCATGGGATTATAAGGAGATGATGATAAGGGATCCACTGAGCGCGAGAACAAT-3′TG (the NcoI site in bold and the FLAG coding sequence underlined) in combination with D14εRev: 5′-GCGGCCGCTTACGACACGTCCTGATC-3′ (the NotI site is in bold) and D14ζForw: 5′CCATGGGATTATAAGGATGATGATGATAAGGGATCCTCGACAGTCGATAAGGAAG-3′TG (the NcoI site is in bold and the FLAG coding sequence underlined) in combination with D14ζRev: 5′GCGGCCGCTTAGTTGTCGCCGCCCTC-3′ (the NotI site is in bold). Amplified fragments were cloned in the NcoI/NotI-digested pγGFPa vector For FLAG-tagged Drosophila, the coding sequence of the 6XHis-g14-3-3 from plasmid pYAHis_g14-3-3 5′GCGCCCCAGATTTTAGAATTCATGGCTAGAGGA-3′ (introduced EcoRI site is in bold) and g14-3-3 rev: 5′-TCGAGTCGACCCGGTCTAGACTACTACTTCTC-3′ (introduced XbaI restriction site is in bold). EcoRI/XbaI-digested PCR fragment was cloned in the EcoRI/XbaI-digested pUAST vector To generate transgenes for Giardia proteins were prepared from 2×109 trophozoites or encysting parasites as previously described Soluble For affinity purifications and mass spectrometry analysis, soluble proteins were obtained from 30–40 flies chilled at 4°C for 1 h, then frozen at -20°C followed by homogenization in 1 ml EB , supplemented with protease and phosphatase inhibitor cocktails (Sigma), then sonicated and the lysate treated as described above.For immunoblot and cross-linking, 1–5 whole flies were homogenized in 50 µl homogenization buffer  supplemented with a protease and phosphatase inhibitor cocktails (Sigma). Protein concentration was quantified by Quant-iT (Molecular Probes).Western blots. Proteins were separated on SDS-PAGE and transferred onto PVDF membrane as described E. coli by affinity chromatography on glutathione-sepharose 4B  and eluted with 10 mM glutathione (pH 8.0) as described by the manufacturer.GST and GST-Difopein were purified from Giardia or Drosophila on GST-difopein, 3 mg of soluble proteins were incubated with 15 µg of glutathione-sepharose immobilized GST or GST-difopein in HT buffer at 4°C for 2 h. After extensive washes with HT buffer, the 14-3-3 proteins were eluted with 100 µl of 2 mM A8Ap synthetic phosphopeptide  reproducing a 14-3-3 binding motif To purify 14-3-3 proteins from Giardia soluble proteins from control WB-C6 strain and transgenic lines were incubated with anti-FLAG beads at 4°C for 3 h and washed with 100 bed volumes of K-HT buffer. To selectively remove proteins bound to 14-3-3, the resin was incubated with 2 mM A8Ap synthetic phosphopeptide, at 4°C for 1 h. Finally, FLAG-fusion proteins were eluted from the resin by incubation with 200 µM synthetic FLAG-peptide at 4°C for 1 h and stored at −70°C until use.FLAG-tagged proteins were purified using anti-FLAG M2 mAb bound to agarose beads (Sigma-Aldrich) per manufacturer’s instructions. An equal amount of His-tagged protein, 1 mg of Drosophila soluble proteins was incubated with 120 µl pre-cleared Ni beads  according to manufacturer, for 4 h at 4°C, followed by multiple washes in lysis buffer with 10 mM imidazole. Bead-bound proteins were recovered by addition of one volume of Laemmli buffer and heating at 95°C for 5 min.For purification of Drosophila protein lysate on Ni-beads as described above. Cross-linking was attained by incubating lysates in 3 mM BS3 (Pierce) as described previously For cross-linking experiments, 6xHis-g14-3-3 was purified from 1 mg of Giardia, trophozoites or encysting cells were prepared as described previously For cell counting, parasites cultured in encysting medium for 12 h were stained with Cy3-conjugated anti-FLAG mAb, FITC-conjugated anti-CWP mAb and DAPI. For each transgenic parasite line, the total amount of cells spotted on the glass-slide and positive for the anti-FLAG staining were counted. The percentage of trophozoites, encysting parasites and cysts (distinguished from trophozoites by co-staining with anti-CWP mAb) was quantified and the mean and standard error of three independent experiments calculated.Drosophila tissues were fixed for 20 minutes in 4% PFA in PBS , blocked for 1 hr at RT in 10% Normal Goat Serum in PBHT . Incubation with the primary antibodies anti-Elav and anti-His at (1∶50) was carried overnight at 4°C. Appropriate Alexa Fluor (Molecular Probes) secondary antibodies were used (1∶500) for 4 hr at RT. Confocal images were obtained with a Biorad Radiance 2100 system.3CN, 1% orthophosphoric acid was used as matrix. Peptides were measured in the mass range of 750 to 4000 Da and all spectra were internally calibrated and processed via the Data Explorer software using ions coming from trypsin autodigestion of FLAG-g14-3-3.Aliquots of affinity purified 14-3-3s (with either anti-FLAG or GST-difopein) were separated on a 1D-gel NuPAGE 4–12%  run in morpholinepropanesulfoninic acid (MOPS) buffer and stained with the Colloidal Blue Staining kit (Invitrogen). Slices were excised and digested with modified sequencing-grade trypsin (Promega) according to published protocols http://www.ebi.ac.uk/clustalw/.Multiple alignments were performed using the Clustal W program at Giardia g14-3-3 with Drosophila D14-3-3ε and LeoII, revealed that all three proteins contain the highly invariant domains typical of all 14-3-3s Giardia protein in Drosophila and the fly proteins in the protozoan.Sequence alignment of Drosophila 14-3-3s were introduced in Giardia by cloning D14-3-3ε and leoII cDNAs Giardia glutamate dehydrogenase promoter ensuring stable mid range expression in all stages, except for a small decrease during encystation Drosophila was achieved under the β-Tubulin-Gal4 (TubG4) enhancer Giardia protein was expressed at relatively high levels and exhibited the expected molecular weight. Importantly, the polyclonal antibodies against D14-3-3ε and Leo did not cross-react with the Giardia protein, which is consistent with the differences in primary sequence and facilitates differentiation between the three proteins when co-expressed. The 3 Leo species resolved into two bands in this particular gel system as reported before Ubiquitous expression of transgenic 6XHis-g14-3-3 in Drosophila proteins in Giardia trophozoites and parasites undergoing encystation. Encystation was monitored with an antibody against Giardia cyst wall protein (CWP) Because g14-3-3 polyglycylation has been linked to its nuclear localization Drosophila cells as in Giardia trophozoites.The subcellular localization of 6xHis-g14-3-3 under the neuronal-specific ElavG4 driver was examined in fly embryonic and adult neurons selected for their accessibility. In comparison to the nuclear Elav protein Giardia and transgenic Drosophila, 14-3-3s from different species co-exist. Because g14-3-3 is the sole isoform in Giardia, it normally forms homodimers. Heterodimer formation with either fly 14-3-3 would provide significant insight into their structural properties. Therefore, FLAG-tagged proteins were purified from Giardia trophozoites by affinity chromatography as detailed in experimental procedures. Tagged 14-3-3s were present only in the transfectant FLAG-eluted fractions, but not in controls  or throughout the adult fly (TubG4). Interestingly, His-g14-3-3 appeared to interact with one of the Leo species resolved (Giardia and Drosophila proteins. These dimers did not contain detectable D14-3-3ε (data not shown). Collectively, these results suggest that g14-3-3 heterodimerizes preferentially with Leo either in Giardia or Drosophila, a property consistent with a 14-3-3ε-like protein.Similarly, nickel affinity chromatography of 6XHis-g14-3-3 from transgenic D14-3-3ε , irresperesolved , lane 6,resolved . To asceGiardia g14-3-3 is constitutively phosphorylated at Thr214 and polyglycylated at Glu246214 of g14-3-3 are well conserved in Drosophila 14-3-3s . Furthermore, inspection of the mass spectrum at high m/z values within the 2586–3572 mass range revealed the peculiar polyglycylation pattern profile  associated with peptide 230DNLNLWVTDSAGDDNAEEK248 (predicted MH+ = 2104.92) To ascertain that D14-3-3ε was modified in EESYK219  exclusiv197AAFDDAIAELDTLS210EESYK216 in the un-phosphorylated (MH+ = 2087.9) and phosphorylated version (MH+ = 2167.9), suggesting that a fraction of FLAG-D14-3-3ε is phosphorylated likely on Ser210 corresponding to Thr214 in g14-3-3 , but in addition a pattern of peaks signifying addition of 7–8 glycines (249 (+ = 1484.6), with the only possible phosphorylation being obligatorily on Ser260   , indicatGiardia, we asked whether 6xHis-g14-3-3 might also be post-transnationally modified in Drosophila. Therefore, whole fly lysates from controls and transgenics expressing 6xHis-g14-3-3 were purified by affinity chromatography on GST-difopein. As described before Given that D14-3-3ε is modified in 202AFDAAITDLDKLTEESYK219 at MH+ = 2029.9  and 249EQIQDVEDQDVS260 (MH+ = 1404.6). Conversely, peptides from 14-3-3ζ were 197QAFDDAIAELDTLNEDSYK215 (MH+ = 2157.98) and 226DNLTLWTSDTQGDEAEPQEGGDN248 (MH+ = 2492.03) . The same result was obtained with proteins purified from transgenic flies, where in addition to the endogenous 14-3-3s, 6xHis-g14-3-3 was also successfully identified (data not shown). The mass spectrum of 6xHis-g14-3-3 only revealed peaks corresponding to the unmodified peptides = 2029.9 , left an 2104.92 , right. D14-3-3ε , the rel2492.03) . FurtherDrosophila does not appear detrimental Giardia has been reported to affect encystation Over-expression of wild type D14-3-3ε, or Leo isoforms in Giardia expressing FLAG-g14-3-3 . Thus, as indicated by their sequence divergence, g14-3-3 is not functionally homologous to Leo.Flies expressing 6xHis-g14-3-3 pan-neuronally under ElavG4, or ubiquitously with TubG4 seemed to develop normally and yield adults of the expected numbers without apparent morphological aberrations. Furthermore, they exhibited grossly normal behaviours and were not deficient in olfactory associative learning (data not shown). This lack of deficits prompted use of 6xHis-g14-3-3-expressing flies to complement loss of function mutations in Leo or D14-3-3ε. Loss of Leo is lethal in Drosophila and homozygotes for the null transposon mutation leo, 40% of the expected D14-3-3ε null homozygotes are recovered (D14-3-3ε null homozygotes (ex4D14-3-3ε). Surprisingly, pan-neuronal (not shown), or ubiquitous 6xHis-g14-3-3 expression enhanced the lethality of ex4D14-3-3ε homozygotes to nearly 100%  remained, Leo levels were significantly elevated in ex4D14-3-3ε null homozygotes as expected Drosophila g14-3-3 may be recognized as an endogenous 14-3-3 thereby negating the normal homeostatic response to D14-3-3ε loss ex4D14-3-3ε nulls. This in turn confirms that the post-translational modifications of g14-3-3 are essential for the functionality of the Giardia protein Because 6xHis-g14-3-3 expression does not affect wild type fly development, it cannot be argued that the enhanced lethality results from interference with D14-3-3ε or Leo-interacting proteins, essential for embryonic development. An alternative explanation is that g14-3-3 interferes with, or negates the compensation by Leo over-expression in the null embryos Giardia is suggested to represent an early branching eukaryote Our data clearly support the proposal Giardia trophozoites, in agreement with the suggested role of the polyglycine chain in preventing g14-3-3 nucleo-cytoplasmic shuttling. In contrast, D14-3-3ε was not found in the nuclei of encysting parasites despite the presence of a short polyglycine chain. This suggests that polyglycine chain length may be critically important for nuclear import factor interaction and its reduced length on D14-3-3ε prohibits translocation. Alternatively, putative import factors are required in addition to length-independent polyglycine chain recognition and they fail to recognize features on D14-3-3ε conducive to nuclear localization. In fact, only a fraction of D14-3-3ε is phosphorylated in the peptide Ala197-Lys216 and phosphorylation of this region has been proven critical for g14-3-3 function, at least during encystation Giardia, functionally confirms the importance of its sequence divergence surrounding the post-translational modification sites on g14-3-3. In agreement with this notion, LeoII (and LeoI and LeoIII) being more divergent in these sequences is not modified at all and as the polyglycylation defective E246A g14-3-3 mutant Giardia nuclei stage-independently. Moreover, the post-translational modifications on g14-3-3 and the consequent changes in subcellular localization may reflect an adaptation similar to that used by Drosophila to increase the functional 14-3-3ζ isoforms by tissue specific alternative splicing.As for the endogenous protein, polyglycylation appears sufficient to exclude D14-3-3ε from the nuclei of Drosophila and Giardia, post-translational modifications are not required or affect the process. Interestingly, although g14-3-3 must be homodimeric in Giardia with such homodimers also observed in Drosophila, it heterodimerizes preferentially with LeoII in both systems. Because D14-3-3ε/g14-3-3 heterodimers were not detected in Giardia or Drosophila, g14-3-3 homodimers and LeoII/g14-3-3 heterodimers are likely more stable and hence favoured over D14-3-3ε/g14-3-3. Homodimerization of human 14-3-3ζ is primarily mediated by three salt bridges, Arg18-Glu89, Glu5-Lys74, and Asp21-Lys85 , and the corresponding residues are partially conserved in LeoII . In human 14-3-3ε and in D14-3-3ε, only the first salt bridge can be formed (corresponding to Arg19-Glu92 in D14-3-3ε), whereas the other residues are substituted in D14-3-3ε by Asn6-Glu77 and Glu22-Gln88. Intriguingly, despite the overall sequence homology with the epsilon subgroup, homodimerization of g14-3-3 may be stabilized by three salt bridges formed between Arg22-Glu97, Asp9-Lys82, and Glu25-Lys93 , similarly to 14-3-3ζ. Consistent with our observations then, LeoII/g14-3-3 heterodimers may be stabilized by gAsp9-dArg77, gGlu25-dArg88, gArg22-dGlu92 (where g indicates Giardia and d Drosophila) salt bridges. In contrast, the D14-3-3ε/g14-3-3 heterodimer can be stabilized only by the lone salt bridge, gArg22-dGlu92, whereas gAsp9-dGlu77 may cause charge repulsion between the two molecules. Because g14-3-3 can form multiple salt bridges presumably to stabilize its own homodimers in a manner akin to 14-3-3ζ, it appears to possess features of both epsilon and zeta isoforms. The later is consistent with the notion that Giardia may be a derived rather than primitive organism.Because preferential g14-3-3/LeoII heterodimerization occurs in both 214 phosphorylation of g14-3-3 in the parasite is currently unknown. Hence we cannot determine whether failure to phosphorylate g14-3-3 in flies is due to absence of a functional homolog. However, g14-3-3 polyglycylation in Giardia is performed by the bifunctional enzyme gTTLL3, a member of the tubulin tyrosine ligase-like family Drosophila possesses polyglycylated microtubules Drosophila, DmTTLL3A mono- and polyglycylates the α- and the β-tubulin, whereas DmTTLL3B mono- and polyglycylates non-tubulin proteins The kinase responsible for ThrDrosophila results from distinct spatial distributions with DmTTLL3s. D14-3-3ε is present in all Drosophila developmental stages and tissues tested Drosophila enzymes. Because polyglycylation is not evident in the fly 14-3-3s, this mechanism of g14-3-3 compartmentalization and stage-specific functions in Giardia does not seem to be utilized in Drosophila. Therefore, a major functional modification of g14-3-3 in the parasite is absent from its Drosophila homolog.It is unlikely that lack of D14-3-3ε and g14-3-3 polyglycylation in D14-3-3ε nulls is probably a consequence of its recognition as an endogenous 14-3-3 protein by the cellular mechanisms responsible for 14-3-3 homeostasis D14-3-3ε nulls, negate the homeostatic response. The lack of post-translational modifications and/or aberrant folding potentially rendering g14-3-3 non-functional in Drosophila, suggest that g14-3-3/Leo heterodimers may also contribute to the dominant negative effects of the Giardia protein in the fly embryos.Our date indicate that despite the similarity of the two proteins, g14-3-3 is not a functional ortholog of D14-3-3ε, at least for processes essential for embryonic viability. The dominant negative effect of g14-3-3 expression in Giardia, may represent an ancestral 14-3-3 containing features common to different subgroups of 14-3-3 and that gene duplication and divergence events have subdivided specific 14-3-3 functions, residing in single of a few amino-acid changes, giving rise to the multimember 14-3-3 families in vertebrates and plants.In conclusion, our data indicate that despite the high level of sequence conservation and structural homology among eukaryotic 14-3-3 proteins, functionality and specificity likely reside in post-translational modifications and particular amino-acids as previously suggested


Psychiatric rehabilitation is an important component in the management of the mentally ill. This article presents a selective review of the publications in this journal. Questions addressed in this review range from assessment of rehabilitation needs to different rehabilitative approaches. Although the number of publications providing the answers is meager, there are innovative initiatives. There is a need for mental health professionals to publish the models they follow across the country. Psychosocial rehabilitation is a therapeutic approach that encourages a mentally ill person to develop his or her fullest capacities through learning and environmental support.Psychiatric rehabilitation and psychiatric treatment are separate, yet equally important complementary components of mental healthcare. Even as psychiatric treatment  aims at controlling psychiatric symptoms, psychiatric rehabilitation focuses on functioning and role outcomes. The new focus of rehabilitation is on wellness and optimum quality of life.The rehabilitation program should start right from the first time the patient has come into contact with a mental health professional. A clinician waiting to start rehabilitation after the patient becomes asymptomatic, may not benefit the patient or the family in the long run.This article reviews publications in the IJP from its inception to date, in the area of rehabilitation. We have tried to summarize these articles and suggest future directions. This includes editorials, commentaries, review articles, book reviews, and case reports.et al.,[The first available publication regarding rehabilitation in our research is by Gupta et al., from therestitutio ad integrum cannot be expected even after the most efficient treatment, although, functioning can fortunately be restored after the disease. He has reported from a community-based mental health review in Sarawak, a state in the federation of Malaysia. They focused on patients in terms of rehabilitation of their previous working capacity. The rehabilitation status was measured by taking into consideration whether they were, ‘working’ ‘probably working,’ ‘Does some work,’ ‘Probably not working,’ and ‘Not working’. They assessed 584 consecutive patients visiting the clinic for follow-up and found that 82% of them were within the first three categories  and were functioning well, while 18% (‘Probably not working’ and ‘not working’) were not functioning, and therefore, could not claim to have been rehabilitated.Schmidt in his aet al,[Nagaswamy et al, carried Channabasavanna, in his eVerma and Shiv reportedGopinath and Rao in theiret al.,[Kastrup et al., have stuet al.,[Mathai et al., in an unAgarwal in his ret al.,[Ponnuchamy et al., examinedThara in a comet al.,[Kumar et al., have assSuresh Kumar observedAlthough rehabilitation of mentally ill is an essential component of psychiatric management there are very few publications in the Indian Journal of Psychiatry in this area. Although many NGOs are providing services in the area of rehabilitation, the models they follow and clinical benefits of the same are not published for the benefit of the psychiatric community. It is important that such rehabilitation service providers are encouraged to publish their activities. It is heartening that following the effort of the IPS to assess the disability in the mentally ill objectively, the government has extended disability benefits to psychiatric patients too. The reach of these benefits to the needy and their social consequences are yet to be assessed. Of late, there has been an initiative from the government of Karnataka to provide short-term residential care for recovering psychiatric patients. The effectiveness of such programs has to be further explored.Also the heterogeneous population of India will have multiple unique needs and problems to be addressed. Although all mental health professionals stress the importance of culture-specific rehabilitative measures, the solution provided by them has not been published.There is a gross scarcity of publications in the IJP with respect to the rehabilitation in psychiatry. The mental health professionals need to take lead in publishing rehabilitation efforts or models being practiced by them in different parts of India. The effect of the disability benefits provided by the government needs to be assessed. The indigenous, innovative models followed need to be published.