The origin of eukaryotes is a fundamental, forbidding evolutionary puzzle. Comparative genomic analysis clearly shows that the last eukaryotic common ancestor (LECA) possessed most of the signature complex features of modern eukaryotic cells, in particular the mitochondria, the endomembrane system including the nucleus, an advanced cytoskeleton and the ubiquitin network. Numerous duplications of ancestral genes, e.g. DNA polymerases, RNA polymerases and proteasome subunits, also can be traced back to the LECA. Thus, the LECA was not a primitive organism and its emergence must have resulted from extensive evolution towards cellular complexity. However, the scenario of eukaryogenesis, and in particular the relationship between endosymbiosis and the origin of eukaryotes, is far from being clear. Four recent developments provide new clues to the likely routes of eukaryogenesis. First, evolutionary reconstructions suggest complex ancestors for most of the major groups of archaea, with the subsequent evolution dominated by gene loss. Second, homologues of signature eukaryotic proteins, such as actin and tubulin that form the core of the cytoskeleton or the ubiquitin system, have been detected in diverse archaea. The discovery of this ‘dispersed eukaryome’ implies that the archaeal ancestor of eukaryotes was a complex cell that might have been capable of a primitive form of phagocytosis and thus conducive to endosymbiont capture. Third, phylogenomic analyses converge on the origin of most eukaryotic genes of archaeal descent from within the archaeal evolutionary tree, specifically, the TACK superphylum. Fourth, evidence has been presented that the origin of the major archaeal phyla involved massive acquisition of bacterial genes. Taken together, these findings make the symbiogenetic scenario for the origin of eukaryotes considerably more plausible and the origin of the organizational complexity of eukaryotic cells more readily explainable than they appeared until recently. Compared to archaea and bacteria , eukaryotic cells are three to four orders of magnitude larger in volume and display a qualitatively higher level of complexity of intracellular organization –3. UnlikThe gulf between the cellular organizations of eukaryotes and prokaryotes is all the more striking because no intermediates have been found. Comparative analysis of eukaryotic cells and genomes confidently maps highly advanced functional systems and macromolecular complexes to the last eukaryotic common ancestor (LECA). The actin and tubulin cytoskeletons, the nuclear pore, the spliceosome, the proteasome and the ubiquitin signalling system are only a few of the striking examples of the organizational complexity that seems to be a ‘birthright’ of eukaryotic cells –10. The Molecular phylogenetics and phylogenomics revealed fundamental aspects of the origin of eukaryotes. The ‘standard model’ of molecular evolution, derived primarily from the classic phylogenetic analysis of 16S RNA by Woese and co-workers and supported by subsequent phylogenetic analyses of universal genes, identifies eukaryotes as the sister group of archaea, to the exclusion of bacteria –16. Withα-proteobacterial endosymbiosis antedated the LECA [However, advances of comparative genomics jointly with discoveries of cell biology have put the archezoan scenario of eukaryogenesis into serious doubt. First, it has been shown that all the purported archezoa possess organelles, such as hydrogenosomes and mitosomes, that appeared to be derivatives of the mitochondria. These mitochondria-like organelles typically lack genomes but contain proteins encoded by genes of apparent bacterial origin that encode homologous mitochondrial proteins in other eukaryotes ,22. Combthe LECA . Compatithe LECA –27. Thesthe LECA ,29. A coThe radical alternative to the elusive archezoa is offered by symbiogenetic scenarios of eukaryogenesis according to which archezoa, i.e. primary amitochondrial eukaryotes, have never existed, and the eukaryotic cell is the product of a symbiosis between two prokaryotes ,23,30,31α-proteobacterium by an archaeon [Although several symbiogenetic scenarios that differ in terms of the proposed partners and even the number of symbiotic events involved have been proposed, the simplest, parsimonious one that accounts for both the ancestral presence of mitochondria in eukaryotes and the hybrid composition of the eukaryotic gene complement involves engulfment of an archaeon ,30,33,36archaeon . Subsequarchaeon ,2,31.A major problem faced by this scenario  is the mechanistic difficulty of the engulfment of one prokaryotic cell by another ,28,29,38However, comparative analysis of the increasingly diverse collection of archaeal and bacterial genomes has yielded multiple lines of evidence that might change the notion of the implausibility of an archaeo-bacterial endosymbiosis. In this article, I discuss the results of genome evolution reconstructions that imply complex ancestral archaeal forma and the discovery of the dispersed archaeal ‘eukaryome’. The eukaryome consists of multiple genes identified in different archaea that encode key components of the cytoskeleton, the cell division apparatus, the ubiquitin system and other signature eukaryotic cellular systems. A complementary line of recent developments shows that massive acquisition of bacterial genes probably occurred on multiple occasions in the course of the evolution of archaea. Taken together, these findings seem to be making the scenario of archaeo-bacterial symbiosis considerably more plausible than it appeared even recently.2.As pointed out above, the ‘standard model’ phylogeny of Woese and co-workers clearly identifies archaea and eukaryotes as sister groups –15. HoweThe eocytes (Crenarchaeota) are not the only group of archaea that has been proposed for the role of the archaeal ancestor of eukaryotes. Evolutionary scenarios based on different versions of metabolic cooperation between the archaeal and bacterial partners of the primary ensodymbiosis, such as the hydrogen hypothesis  and the Thermoplasma or a related, wall-less archaeon also has been proposed as a plausible ancestor of eukaryotes, on the basis of biochemical and cytological considerations, long before the phylogenomic analysis became feasible [Most of the inferences of archaeo-eukaryotic relationship studies mentioned above were based either on phylogenetic analysis of a single, universal gene, such as 16S rRNA, or on concatenated sequences of several universal proteins , or on non-sequence characters such as gene repertoires (phyletic patterns) and domain architectures of multidomain proteins. Obviously, extensive sequencing of genomes from all three domains of cellular life opens the door for comprehensive phylogenomic analyses. The first such extensive phylogenomic exercise involved analysis of nearly 6000 gene sets from 185 archaeal, bacterial and eukaryotic genomes and employed a supertree approach to combine information from the multiple trees; the results suggested affinity between eukaryotes and the Thermoplasmatales branch of Euryarchaeota, albeit with limited statistical support . Thermopfeasible –61.Another comprehensive phylogenetic analysis of individual eukaryotic genes of apparent archaeal origin has suggested an origin outside of the known archaeal diversity for most of these genes but also identified many genes with a crenarchaeal (eocyte) affinity and a smaller number of genes with a euryarchaeal affinity . In thisCandidatus Caldiarchaeum subterraneum has suggested yet another archaeal phylum, dubbed Aigarchaeota [The diversity of the outcomes of phylogenetic analysis, with the origin of eukaryotes scattered around the archaeal diversity, has led to considerable frustration and suggested that a ‘phylogenomic impasse’ has been reached, owing to the inadequacy of the available phylogenetic methods for disambiguating deep relationships . Howeverrchaeota . The latrchaeota ,72–74 The indispensable structural framework of all eukaryotic cells is the advanced, elaborate cytoskeleton that consists of two major types of elements, namely actin-based filaments and tubulin-based microtubules ,103. TheKorarchaeum and Ca. Caldiarchaeum subterraneum, with the implication that actin was already present in the last common ancestor of the TACK superphylum [Recently, analysis of the expanding archaeal genome collection has changed this perspective. Proteins with high sequence similarity and unambiguous phylogenetic affinity to eukaryotic actins have been discovered in the crenarchaeal order of Thermoproteales, erphylum ,111. Folerphylum –114.Sequence analysis of the crenactins has indicated that these proteins contained several insert that are present in eukaryotic actin-related proteins (ARPs) but not in actins themselves . Once twNitrosoarchaeum and Nitrosotenius [Highly conserved orthologues of tubulins, named artubulins, so far have been discovered only in the genomes of several ammonium-oxidizing Thaumarchaeota of the genera sotenius ,119. Altsotenius . The strThus, in a remarkable departure from recent common beliefs, both major forms of the eukaryotic cytoskeleton seem to belong within the archaeal heritage of eukaryotes. However, the apparent ancestral forms have been detected in widely different groups of extant archaea.(b)Korarchaeum, with the implication that the division mechanisms of these archaea are similar to the bacterial one [Cell division obviously is central to all cellular life forms. Nevertheless, the cell division mechanisms substantially differ between bacteria and at least some archaea, on the one hand, and eukaryotes, on the other hand. In bacteria, division is coupled to chromosome replication, with the progeny DNA molecules being pumped into the daughter cells concomitant with replication. Division is then completed by septation, with the formation of the septum driven by the Z-ring that consists of the FtsZ protein, a GTPase that is a distant homologue of eukaryotic tubulins ,121. In rial one .Sulfolobus acidocaldarius [Sulfolobales and Desulfurococcales, as well as some Thaumarchaeota and Euryarchaeota [Nitrosopumilus maritimus [Sulfolobus acidocaldarius, has been shown to form helical filaments that mediate membrane scission during cell division [Unexpectedly, a distinct division system homologous to the eukaryotic ESCRT-III membrane remodelling complex has been discovered in the crenarchaeon aldarius –125 and rchaeota . Subsequaritimus . Furtherdivision . This fiPicrophilus torridus , lacks all three division machineries discussed above and hence is expected to employ a novel mechanism [The presence of the FtsZ-based and ESCRT-III-like division systems in a broad variety of diverse archaea implies that both machineries were present in the LACA, with subsequent differential losses in multiple lineages. The Crenarchaeota in the order Thermoproteales lack both of these division systems and thus must employ a distinct third one that most probably relies on the crenactin cytoskeleton ,113. Givechanism .(c)The ubiquitin (Ub) system is the central signalling and regulatory network of the eukaryotic cell. This extremely complex machinery regulates protein degradation, topogenesis and function in all eukaryotes through modification of proteins by conjugation with various forms of (poly)Ub and its paralogues –130. ForCa. Caldiarchaeum subterraneum, the founding member of the putative phylum Aigarchaeota, has transformed the entire story of the origin of the Ub system by revealing a predicted operon encoding a Ub-like protein and homologs of all three Ub ligase subunits along with a key deubiquitinating enzyme [Ca. Caldiarchaeum subterraneum show high sequence similarity to eukaryotic counterparts, and phylogenetic analysis of the E1 subunit of Ub ligase and the deubuiqitinating enzyme MPN  place Ca. Caldiarchaeum subterraneum in the midst of eukaryotes [Analysis of the genome of g enzyme . Operonsg enzyme . In contkaryotes . Subsequkaryotes . The posOther homologues of the Ub system components are scattered among archaea ,137. In (d)The RNAi is a hallmark eukaryotic functional system that is involved in the defence against viruses and transposons and in multiple pathways of gene expression regulation –142. PhyThe Dicers are signature eukaryotic proteins that have no direct counterparts in bacteria or archaea and encompass a fusion of a helicase domain and two RNAse III domains that are unique to eukaryotes. The dsRNA-specific RNAse III is nearly ubiquitous in bacteria and apparently has been acquired by some mesophilic Euryarchaeota via HGT . By contIn contrast to Dicers, the Argonautes have numerous homologues in bacteria and archaea, primarily Euryarchaeota, and phylogenetic analysis clearly points to a euryarchaeal origin of the eukaryotic Argonaute family ,153. ComA third key component of the eukaryotic RNAi is an RNA-dependent RNA polymerase (lost in some eukaryotic lineages including vertebrates) that serves as an amplifier of siRNAs. Homologues and possible ancestors of this polymerase have been identified in some bacteriophages where they are most likely to function in transcription, as DNA-dependent RNA polymerases ,157.Thus, the RNAi system, a signature eukaryotic defence and regulatory network, appears to have evolved from the archaeal Argonaute-centred defence machinery through the accretion of additional components. The ancestral archaeal system remains to be thoroughly characterized, and it cannot be ruled out that functional analogues and possibly even direct homologues of Dicers will eventually be discovered in some archaea.5.Arguably, the greatest difficulty faced by the endosymbiotic scenarios of eukaryogenesis is the apparent implausibility (or at least extreme rarity) of the engulfment of one prokaryotic cell by another. The recent advances of comparative genomics, complemented by the progress in the cell biology of archaea, seem to be closing this gap. Combined with the quantitative findings of genome evolution reconstructions on extensive differential gene loss in most archaeal lineages, the discovery of the ‘dispersed’ archaeal eukaryome implies a highly complex archaeal ancestor of eukaryotes ,86. ConcMethanosarcinales or Halobacteria, clearly have acquired numerous genes via HGT [α-proteobacterial signal among the eukaryotic genes of apparent bacterial descent [Most probably, the archaeal ancestor of eukaryotes was a wall-less mesophile that coexisted with diverse bacteria, so even a limited capacity for phagocytosis would greatly facilitate the capture of prospective endosymbionts. Extant mesophilic archaea, such as  via HGT ,100,101. via HGT . Conceiv descent ,160.The results of phylogenomic analysis outlined above strongly suggest that the ancestor of eukaryotes was a deep branch within the TACK superphylum ,92, possIf the archaeal ancestor of eukaryotes  was a complex organism with some signature features of eukaryotic cells, such as the cytoskeleton, the question emerges whether this ancestral form was an archaeon or a primitive, amitochondrial eukaryote, i.e. an archezoan. The answer hinges on the definition or rather the salient features that ‘make an organism a eukaryote’. The cytoskeleton, membrane remodelling systems such as ESCRT-III, the Ub system and RNAi certainly are eukaryotic signatures. Yet, the defining traits of eukaryotes appear to be the large size of the eukaryotic cells coupled with the presence of the elaborate endomembrane system which includes the nucleus endowed with nuclear pores and the splicing machinery that is linked to the nucleocytoplasmic trafficking. Quantitative argument has been developed that the eukaryotic cellular organization is unsustainable without multiple energy-converting organelles, such as mitochondria . Further6.Four groups of recent observations increase the plausibility of the symbiogenetic scenario for the origin of eukaryotes. The first line of evidence comes from the reconstructions of archaeal genome evolution which imply complex ancestral forms, with the subsequent evolution in most lineages dominated by gene loss. The related and perhaps most important clues come from the observations on the archaeal eukaryome that is scattered among diverse extant archaea. The putative complex archaeal ancestor of eukaryotes could have encoded most if not all components of the eukaryome within the same genome, possibly endowing this ancestral archaeon with certain eukaryote-like functionalities such as the ability to efficiently engulf other cells (a primitive version of phagocytosis). The third line of evidence consists of the increasingly confident demonstrations of the origin of the core eukaryotic genes from within the archaea, or more specifically, from a deeply branching group within the TACK superphylum. Given that all known extant members of this superphylum are typical archaea and not archezoa, these findings appear to favour an archaeal host for the proto-mitochondrial endosymbiont. Finally, the indications that massive acquisition of bacterial genes most probably triggered the emergence of the major groups of archaea put the origin of eukaryotes into a more general evolutionary context. These discoveries make the origin of eukaryotes appear less dramatically different from the origin of other groups of organisms than is generally perceived. Horizontal transfer of numerous genes appeared to have been central in each case. The key difference is that in eukaryotes the source of the foreign genes, i.e. the endosymbiont, survived as an organelle, precipitating the radical restructuring of the cell. Given the likely origin of eukaryotes from within the archaeal diversity and the observations on the dispersed eukaryome, there seems to be high promise of new evolutionary insights coming from metagenomics and single-cell genomics. The discovery of archaeal descendants of the elusive host of the mitochondrial endosymbiont cannot be ruled out.Shortly after this manuscript was submitted, a game-changing discovery bearing on the archaeal ancestry of eukaryotes has been published ,162. Dee


Objective. The objective of this study was to evaluate the regression of temporomandibular pain as a result of intra-articular injections of platelet-rich plasma (PRP) to patients with temporomandibular joint dysfunction previously subjected to prosthetic treatment. Materials and Methods. The baseline study material consisted of 10 patients, both males and females, aged 28 to 53 years, previously treated due to painful temporomandibular joint dysfunction using occlusal splints. All patients were carried out to a specialist functional assessment of the dysfunction using the Polish version of the RDC/TMD questionnaire axis I and II. Intra-articular injections were preceded by a preparation of PRP. The injection sites were determined by the method used during arthroscopic surgical procedures. Following aspiration, 0.5 mL of plasma was injected into each temporomandibular joint. Results. The comparison of the intensity of pain during all examinations suggests a beneficial effect of the procedure being performed as the mean VAS score was 6.5 at examination I, 2.8 at examination II, and 0.6 at examination III. Conclusion. Application of the intra-articular injections of platelet-rich plasma into the temporomandibular joints has a positive impact on the reduction of the intensity of pain experienced by patients treated for temporomandibular joint dysfunction. A number of conservative methods are used in the treatment of temporomandibular joint dysfunction pain syndrome, including occlusal splints of various designs, supportive physical therapy procedures, rehabilitation involving muscular training, and specialist psychological support –10.Intra-articular administration of medications is an established method of treatment, particularly in orthopedic and rheumatic disorders associated with pain, effusion, inflammation of cartilage, and bone and joint capsules as well as fibrous adhesions. Currently, agents used for intra-articular injection within the temporomandibular joint regions include hyaluronic acid and steroids , 12.Although autogenous platelet-rich plasma (PRP) concentrates were developed in the 1970s, the medical use of these materials was facilitated only by subsequent technological advances, particularly by progress in medical instrumentation. In 1997, Whitman et al. were the first to present a method for preparation and use of platelet-rich plasma to accelerate healing processes. Centrifugation of full autogenous blood yields a platelet concentrate with platelet concentration of ca. 1 million cells per cubic millimeter of plasma. The concentrate is produced in the process of collecting peripheral vein blood (most commonly from the ulnar vein) directly from patients, with blood being centrifuged during the procedure. To date, platelets were demonstrated to contain more than 30 growth factors, the most important of them being the platelet derived growth factor (PDGF), transforming growth factor (TGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), and vasoendothelial growth factor (VEGF) –21.Platelets are anuclear, flat disc-shaped cells formed in bone marrow; about 30% of all platelets are found in the spleen. The life span of platelet cells ranges from 8 to 12 days; cells are eliminated by the mononuclear phagocyte system. When in the interphase, platelets have no thrombogenic potential; thrombin is required for their activation which involves a change in the shape and the release of granules. The main function of the platelets is the control of hemostasis. Platelets form hemostatic plugs at injury sites, participating in blood coagulation processes. Wound healing starts with platelet thrombus formation, platelet degranulation, and the release of growth factors. The next stage involves fibroplasia and angiogenesis followed by the synthesis of structural connective tissue elements. This leads to increased resistance to wound disruption and then to scar remodeling. New collagen is generated owing to chemotactic and mitogenic effects of FGF on fibroblasts, stimulating proliferation and differentiation of keratinocytes thanks to the presence of highly concentrated growth factors , 18.Platelet-derived growth factors contained in the concentrate are peptides that stimulate proliferation, differentiation, and migration of cells. These properties determine the usefulness of growth factors in the healing process. Most growth factors present within the platelet-rich plasma have mitogenic properties leading to an increase in the repair cell counts. Growth factors derived from centrifuged blood were used for the first time by Knighton in patients with chronic skin ulcerations .Platelet-rich plasma contains proteins that are responsible for cell adhesion, namely, fibrin, fibronectin, and vitronectin. It stimulates tissue regeneration processes by stimulating fibroblasts to produce structural proteins for use in formation of new collagen and elastin, support of remodeling and angiogenesis (formation of new vessels), and activation of mesenchymal stem cells. PRP is widely used in surgical procedures, in the treatment of burns and extensive, difficult-healing wounds, orthopedic ligament injuries, and connective tissue injuries. An additional advantage of this method is the safety of the material and a zero possibility of hypersensitivity to injected plasma. It should be kept in mind that blood and plasma may be carriers of pathogens such as HIV, CMV, EBV, or HCV, and therefore precautions should be used consisting of appropriate staff apparel and patient behavior , 21, 22.The objective of this study was to evaluate the regression of temporomandibular pain as a result of intra-articular injections of platelet-rich plasma to patients with temporomandibular joint dysfunction previously subjected to prosthetic treatment.The baseline study material consisted of 10 patients, both males and females, aged 28 to 53 years, previously treated using occlusal splints, who reported at the Consulting Room of Temporomandibular Joint Dysfunctions of the Department of Dental Prosthetics of the Jagiellonian University Medical College in Krakow between 01.09.2013 and 20.05.2014 due to painful dysfunction of the stomatognathic system.All patients were carried out to a specialist functional assessment of the masticatory organ using the Polish version of the RDC/TMD questionnaire axis I and II (examination I). The following parameters were analyzed in detail for the purposes of this study: changes in the intensity of pain as reported by the patients with regard to temporomandibular joints and stomatognathic system muscles, pain intensity characteristics, level of impairment, and grade of chronic pain , 24.The assessment involved also a subjective assessment of pain within the stomatognathic system muscles and temporomandibular joints, evaluation of the range and symmetry of mandible motion, acoustic symptoms within the temporomandibular joints, and the impact of the masticatory organ health on overall well-being , 3, 23.The inclusion criteria included temporomandibular joint dysfunction pain manifested as lack of coordination in operation of articular heads and disc (disc translocation with or without disc blockade/crackling sounds/) and dysfunctions presenting with excessive masticatory muscle tone as the dominant problem and lack of benefits from previous treatment methods. All subjects reported that the pain had sustained for at least 3 months and that they continued the prosthetic treatment.The exclusion criteria included poor overall health, unwillingness to participate, numerous dental defects, diagnosis of a connective tissue disease, and dysfunctions classified as RDC/TMD groups IIIb and IIIc. Contraindications associated with the use of platelet-rich plasma  were also taken into account , 23, 24.Intra-articular injections of platelet-rich plasma were preceded by collection of peripheral blood from the ulnar vein of the patient in accordance witj the procedures in force using single-use, closed vacuum systems and glass tubes with sodium citrate as an anticoagulant . After mThe injection sites were determined by the method used during arthroscopic surgical procedures within the temporomandibular joints. Patients were prepared for the procedure by a line being drawn on their skin between the earlobe and the outer eye corner. Three segments were marked at 10 mm intervals starting from the earlobe. The lengths of the lines were 3 mm (first line), 5 mm (second line), and 7 mm (third line). The platelet-rich plasma injection site was marked by the tip of the third line corresponding to the upper compartment and retrodiscal zone . The skiThe following parameters were assessed in the specialist baseline examination and the follow-up examinations using the RDC questionnaire: the presence and intensity of spontaneous and palpation-induced pain within temporomandibular joints and masticatory muscles, the range and symmetry of mandible motion, abduction trajectory, and the presence of acoustic symptoms within temporomandibular joints during all mandible movements and accompanying symptoms such as squeaking sounds and tinnitus in ears. In one case, classified as RDC group IIb, active unblocking of the articular joint was first performed; the patient was subsequently qualified for platelet-rich plasma injection , 23.The results were subjected to statistical analysis using the basic procedures such as determination of means, standard deviations, medians, and post hoc ANOVA Friedmana statistical significance test.The group of patients qualified for the study consisted of two males and eight females. The mean age was 37.6 years. According to the results of baseline examinations, the patients reported for prosthetic treatment for the following reasons: pain within one or both temporomandibular joints, spontaneous or occurring at mandible movements, chewing, tight jaw occlusion, or joint palpation. The pain was located in the joints or the masticatory muscles, radiating into the temporal and cranial region in 2 cases. Joint crackling sounds were heard upon mandible movements by 4 patients, while a reduced range of mandible abduction was observed in 1 patient. The following diagnoses were made on the basis of RDC/TMD questionnaire axis I survey: 1 case of group I a, 4 cases of group II a, case of group II b, and 4 cases of group III a .The comparison of the intensity of temporomandibular joint and masticatory muscles pain at baseline and follow-up examinations suggests a beneficial effect of the procedure being performed as the mean VAS score was 6.5 at examination I, 2.8 at examination II, and 0.6 at examination III. The result of subsequent investigation differs in the statistically significant way Tables  and 4. IChronic facial pain is a common cause of impairment in the everyday activities of patients and requires numerous therapeutic procedures being initiated by physicians of different dental specialties as well as by neurologists, laryngologists, maxillofacial surgeons, and psychiatrists. This suggests a need for interdisciplinary treatment due to the complex character of complaints. Temporomandibular joint pain is a special problem as the mandible motion upon chewing and talking constitutes a stimulus for a significant increase in the pain being experienced , 31–34.Acoustic symptoms and tenderness on palpation (associated with hyperactivity) of mandible lifting muscles are an evidence of overloads located within the posterior disc ligament . The area is poorly accessible for application of healing-promoting drugs and the physical therapy procedures applied in these cases are not always satisfactory.Many researchers studying PRP treatments highlight the high efficacy of this method of management of musculoarticular disorders and its safety due to the use of autologous material as well as the low costs of treatment , 35–57.Daif compared the efficacy of intra-articular injections within the upper temporomandibular joint compartment and additional injections in the articular capsule region points out the superiority of the latter method for the treatment of dysfunctions involving disc translocation with no disc blocking . PRP is Today, the use of platelet-rich plasma has been expanded no include numerous indications after orthopedic surgeries to treat sports-related injuries. PRP is widely used for tissue healing in many anatomical regions as well as in the treatment of pathological lesions of bone, cartilage, ligament, and muscle tissues. The popularity of this novel treatment method triggered a rapid increase in research. However, differences in application techniques and PRP compositions make comparisons of efficacy results difficult. Potential complications following the procedures involving PRP administration are mild and therefore this method of treatment appears to be safe. It brings various potentially positive effects for the damaged musculoskeletal system tissues. Platelet-rich plasma injection is used with increasing frequency in reconstructive orthopedic procedures , muscle injuries, dental surgeries, and implantations , 45–51. A separate opinion suggesting the lack of confirmed benefits of PRP on musculoarticular tissue healing has been presented by Willits et al., who claim that the review of PubMed and Medline data performed in April 2013 does not warrant any positive opinions on the clinical use of this method .The results of our preliminary research suggest beneficial effects of platelet-rich plasma in intra-articular injections as a supplement to the basic prosthetic treatment of temporomandibular joint dysfunction. The reduction in pain and gradual restoration of functional capabilities of the stomatognathic system as the result of the treatment of interest raises hopes that future studies would lead to establishment of appropriate management algorithms.Additional intra-articular injections of platelet-rich plasma into the temporomandibular joints have a positive impact on the reduction in the intensity of pain experienced by patients treated for temporomandibular joint dysfunction.Determination of appropriate treatment algorithm making use of intra-articular injection in patients with temporomandibular joint dysfunction pain requires further studies and longer follow-up of the study group.Conclusions are as follows.


The etiology and maintenance of insomnia are proposed to be associated with increased cognitive and physiological arousal caused by acute stressors and associated cognitive rumination. A core feature of such hyperarousal theory of insomnia involves increased sensory processing that interferes with the onset and maintenance of sleep. In this work, we collected structural magnetic resonance imaging data from 35 patients with primary insomnia and 35 normal sleepers and applied structural covariance analysis to investigate whether insomnia is associated with disruptions in structural brain networks centered at the sensory regions . As expected, insomnia patients showed increased structural covariance in cortical thickness between sensory and motor regions. We also observed trends of increased covariance between sensory regions and the default-mode network, and the salience network regions, and trends of decreased covariance between sensory regions and the frontoparietal working memory network regions, in insomnia patients. The observed changes in structural covariance tended to correlated with poor sleep quality. Our findings support previous functional neuroimaging studies and provide novel insights into variations in brain network configuration that may be involved in the pathophysiology of insomnia. Chronic insomnia is the most prevalent sleep complaint, which affects about 20% of adult population worldwide . InsomniIn the past two decades, a number of studies using diverse functional neuroimaging techniques have revealed that central nervous system hyperarousal represents a major pathophysiologic pathway in the development and maintenance of insomnia –16. A coIt has been known that the dynamic emergence of coherent physiological activities that span distinct brain regions making up functional networks is supported by complex structural networks constituted by neuronal elements of the brain . Thus, aIn the present study, the structural covariance of networks involved in sensory processing was investigated by seeding from the primary visual cortex (PVC), the primary auditory cortex (PAC), and the olfactory cortex (OLF). Based on existing functional neuroimaging findings, we hypothesized that patients with PI would show greater sensory-motor connectivity compared with normal sleepers, and such alteration would be correlated with poor sleep quality.This work studied 35 patients with PI  and 35 healthy controls , who were recruited at the Neurology Department of the People's Hospital of Zhengzhou University. All participants were right-handed. Diagnosis was performed according to the DSM-IV inclusion criteria for PI [MRI data acquisition was conducted on a Siemens 3.0 T TrioTim whole-body scanner  using a 12-channel array coil. High-resolution structural volumes were obtained using a T1-weighted 3-Dimensional Magnetization Prepared Rapid Acquisition GRE (3D-MPRAGE) sequence .The T1-weighted MR images were processed with the CIVET MRI analysis pipeline (version 1.1.12)  developehttp://www.math.mcgill.ca/keith/surfstat/) for Matlab .The statistical analysis of SCNs was conducted using the SurfStat toolbox . Prior to the network analysis, a linear regression was performed at every cortical vertex to remove the effects of age and gender. The residuals of this regression were used to substitute for the raw cortical thickness measurements. Then, for each group, the averaged adjusted cortical thickness within each of the seed regions was correlated with all other vertices on the entire cortical surface. Significant correlation was interpreted as connectivity. Following previously reported nomenclature [T at a surface point i was SCNs were constructed by seeding from the primary sensory regions, which were delineated using the Automated Anatomical Labeling (AAL) atlas . These snclature , the mod seed thickness and group, together with a seed thickness × group interaction term. We assessed differences in structural covariance between groups by testing the significance of the interaction term at each vertex [T at a surface point i was To compare the structural covariance in the group of patients with PI and in the group of healthy controls, we used linear interaction models, which contained terms for theh vertex . The mod seed thickness and PSQI score as well as a parametric interaction term for seed thickness × PSQI score. The model fitted for the cortical thickness T at a surface point i was We assessed the modulation of covariance strength by sleep quality that was measured as PSQI score. These models contained terms forP < 0.05. To illustrate trends, surface maps were also shown at an uncorrected threshold of P < 0.05.Random field theory (RFT)  was utilP > 0.05, uncorrected). Patients with PI had significantly higher total PSQI score  relative to healthy controls, illustrating that patients had significantly poorer sleep quality.Demographic features for the groups of patients with PI and healthy controls are shown in P < 0.05, uncorrected) in the left anterior PAC, the left inferior lateral occipital cortex (LOC), the left the paracentral lobule (PCL), the left anterior cingulate cortex (ACC), the right middle cingulate cortex (MCC), the right cuneus, the right PVC, the right precuneus, and the right parahippocampal gyrus (PHG). In addition, patients with PI also tended to have decreased cortical thickness in the lateral prefrontal cortex (PFC) . Furthermore, correlations between the vertex-wise cortical thickness and the sleep quality  are shown in P < 0.05, uncorrected) of thickness with PSQI; the areas tended to have decreased thickness in PI and the left inferior parietal cortex (IPC) and the right opercular part of inferior frontal gyrus (IFGoperc) exhibited trends of negative thickness, PSQI correlations .Between-group differences in cortical thickness are illustrated in P < 0.05, RFT-cluster corrected). The right PVC additionally encompassed the right posterior insula .The SCNs seeded from the left and right PVC (CAL) are illustrated in Figures P < 0.05, RFT-cluster corrected) was similar to that seen in healthy controls, but more widespread in the medial motor areas (the PCL and the supplementary motor areas (SMA)), the medial temporal cortex (PHG), the left postcentral gyrus, and the right insula and less widespread in the LOC and the left vmPFC.In patients with PI, the distribution of significant correlations for PVC  between the bilateral PVC and the medial motor areas (PCL/SMA) and the left cuneus, and between the left PVC and the left dorsal parietooccipital region, the right PHG and the right inferior temporal cortex (ITC), and between the right PVC and the right vmPFC and the right posterior insula, in patients with PI. Patients also showed trends of decreased covariance  between the bilateral PVC and the lateral PFC, the IPC, the LOC, the left superior temporal cortex (STC), the dorsal medial prefrontal cortex (dmPFC), and the IFGoperc.Figures P < 0.05, RFT-cluster corrected). The left PAC network additionally included the left and right inferior PFC ; the right PAC network also included the left and right medial visual areas and the left ITC .Figures P < 0.05, RFT-cluster corrected) in PI. However, in patients, we observed additional correlations of the left PAC with the lateral PFC, the pre- and postcentral gyri, the lateral temporal and parietal areas, the dmPFC, the vmPFC, and the right superior cuneus; and additional correlations of the right PAC with the left postcentral gyrus, the medial occipital cortex, the medial motor areas (PCL/SMA), the precuneus/PCC, the right dmPFC and vmPFC, and the medial and inferior temporal cortex.The left and right PAC were also connected with the bilateral opercular areas , as well as trends of increased correlations with the dorsal and inferior lateral PFC, the bilateral precentral gyri, the right postcentral gyrus, the LTC, the left posterior insula, the lateral parietal cortex, the medial motor areas (PCL/SMA), the dmPFC, the left OLF, the right vmPFC, and the right superior cuneus . Patients' right PAC network showed significantly increased correlation with the right medial motor area (PCL/SMA) , and trends of increased correlations with the left postcentral gyrus, the left medial motor area (PCL/SMA), the left cuneus, the precuneus/PCC, the vmPFC, the right MCC, the right OLF, the right ITC, and the right posterior insula . Furthermore, patients showed trends of decreased correlations  between the bilateral PAC and the lateral PFC and the IFGoperc, and between the left PAC and the right ITC (the fusiform gyrus (FFG)), and between the right PAC and the IPC and the LTC.Figures P < 0.05, RFT-cluster corrected). The right PVC additionally encompassed the precuneus/PCC, the ACC and the MCC, the dmPFC, the left lingual gyrus, the right precentral gyrus, and the right posterior insula .The SCNs seeded from the left and right OLF are illustrated in Figures P < 0.05, RFT-cluster corrected). However, in PI, the left OLF network showed additional correlations in the bilateral insula, the temporoparietal regions, the ACC, the right MCC, and the lower parts of the right pre- and postcentral gyri. The right OLF network in patients showed more widespread correlations with the dorsal central areas, the superior parietal cortex, the medial motor areas (PCL/SMA), the medial occipital lobes, and the right precuneus, whereas the correlations to the left MCC and the left dmPFC did not reach significance .Similar to controls, patients' OLF SCNs also included the anterior and inferior PFC, the parietal cortex, and the vmPFC . Both the left and the right OLF networks in PI also showed trends of increased correlations with the dorsal central areas, the medial motor areas (PCL/SMA), the medial temporal cortex (PHG), and the medial occipital lobes . Additionally, in patients, the left OLF network had trends of increased correlations with the right ACC, the right inferior temporal cortex, the lowermost part of the right postcentral gyrus, the right anterior insula, and the bilateral precuneus/PCC ; the right OLF network had trends of increased correlations with the dorsal parietooccipital regions, the right inferior occipital gyrus, and the right PAC (HES) . Moreover, patients showed decreased correlations  between the bilateral OLF and the lateral PFC, the IPC, the LTC, and the dmPFC, and between the right OLF and the bilateral IFGoperc.Figures P > 0.05, RFT-cluster corrected). Analysis of the uncorrected modulations identified trends of positive and negative modulatory effects of PSQI score on the sensory networks  . In addition, we also found trends of increased covariance of PVC with the medial motor area (PCL/SMA) and the left cuneus, and of PAC with the primary motor and somatosensory cortex , the medial motor area (PCL/SMA), the cuneus visual cortex, and the OLF, and of OLF with the primary motor and somatosensory cortex , the medial motor area (PCL/SMA), the dorsal visual cortex, and the right PAC. The findings are well in line with data from previous functional connectivity analysis. Killgore and colleagues examined resting-state functional connectivity differences between individuals with or without insomnia-related sleep problems [In the current study, compared with the healthy controls, the primary alterations in SCNs in patients with PI were the increased covariance between the sensory and motor regions. Significantly increased covariance was found between the left PAC and the left primary somatosensory cortex , and between the right PAC and the right medial motor area (PCL/SMA), and between the right OLF and the left cuneus . Although there is no full understanding about the functions inherent to DMN, in general, DMN concerns the neural network that is active when the individual is not focused on an external task mobilizing the explicit attentional resources and the brain is at wakeful rest [Furthermore, patients with PI also showed increased structural covariance between the sensory regions and the DMN regions, such as the precuneus/PCC, the vmPFC, the anterior LTC, and the PHG. In particular, the increased structural covariance between the right PAC and the bilateral anterior LTC survived correction for multiple comparisons (ful rest . Studiesful rest . Dysfuncful rest . Drummonful rest  investigful rest  examinedful rest . Alternaful rest .Patients with PI also tended to have increased structural covariance of the sensory regions, especially the left OLF, with the ACC and the insula that are nodes within SN. SN is thought to recruit relevant brain regions for the processing of sensory information and integrate processed sensory data with visceral, autonomic, and hedonic signals in order to guide behavior , 63. AltAnother finding in the current study is that patients with PI showed trends of decreased connectivity of the seeds of primary sensory regions with the frontoparietal working memory network consisting of the lateral PFC, the IFGoperc, and the IPC . Such coWe assessed the relationship between the structural covariance and sleep quality by testing the parametric interaction between the PSQI score and the structural covariance strength from seed regions across all participants. Although all modulation of structural covariance by PSQI did not survive multiple comparison correction, analysis of the uncorrected modulations found trends of positive and negative modulatory effects of PSQI score on the sensory networks; and it is notable that the patterns of the positive and negative modulation trends were, respectively, consistent with the patterns of uncorrected connectivity increase and reduction in PI. This further illustrated that the structural covariance alterations in patients with PI observed in this work may be related to the pathophysiology of insomnia.In this work, we noted some asymmetries between the SCN alteration maps of the left and right sensory regions, for example, the increased structural covariance maps of the left and right PAC. Although, by far, there is no clear evidence about asymmetric effects of insomnia on the brain, previous studies have also reported lateralized patterns of brain structural alterations in PI –69. It iThere are limitations of the current study that should be addressed in future investigations. First, most present results did not survive the correction for multiple comparisons, which might be caused by the relatively small sample size. It is desirable to increase the sample size in the future work to increase the statistical power. Second, although our findings in structural covariance were qualitatively well in line with the data from previous functional connectivity analyses, it is unclear about the quantitative similarity between the observed SCNs and the functional connectivity networks derived from fMRI data. There is still lack of systematic comparison of structural covariance and functional connectivity networks. It should be cautious to generalize the findings of SCNs in PI to other networks. Combinations of multimodal neuroimaging approaches should yield a more comprehensive understanding of how abnormalities in the brain network architecture are associated with functional deficits. Third, the structural network was group-based so that we could not explore the network organization for individual subjects. Furthermore, subcortical regions were not included in this study, as this study concentrated on the structural covariance in cortical thickness. It is desirable to extend the study to subcortical regions to explore their contributions to the disruptions of the SCNs in PI. Some existing studies, that is, , attemptIn summary, we, for the first time, used a seed-based SCN mapping approach to investigate large-scale structural networks in PI. The results demonstrated increased covariance in cortical thickness between sensory and motor regions, and likely between sensory regions and DMN and SN regions, and trends of decreased connectivity between sensory regions and the frontoparietal working memory network regions, in patients with PI. In addition, the observed alterations in structural covariance tended to be correlated with poor sleep quality. Our findings support the aberrant functional activation and connectivity in insomnia reported by previous functional neuroimaging based studies and also provide novel insights into variations in brain network configuration that may be involved in the pathophysiology of insomnia.Supplementary Figure S1 illustrates the anatomical locations of the seed regions for structural covariance network construction, which were delineated using the AAL atlas.


Psoriasis is a chronic inflammatory skin condition with significant physical and psychosocial comorbidity. A workshop of leading experts in dermatology and psychology with the purpose of better understanding the current role of psychological comorbidities in psoriasis was held by the International Psoriasis Council in November 2013. The role of stress reactivity with a focus on the hypothalamic-pituitary-adrenal axis was emphasized. While cognitive behavioral therapy remains the most extensively studied and successful treatment strategy in patients with psoriasis and various psychological comorbidities, new and innovative interventions such as online-based therapies have recently emerged. Strategies and recommendations toward approaching psychological comorbidities are discussed. Psoriasis is a chronic, relapsing and remitting inflammatory disease of the skin with a prevalence of 2–3% worldwide Koo, . ChronicThe International Psoriasis Council (IPC) is a dermatology-led, global non-profit organization dedicated to innovation across the full spectrum of psoriasis through research, education, and treatment. Recently, the IPC has led an initiative to better define the association of various psychological comorbidities with psoriasis. In November 2013, a workshop was held in Boston, MA, which assembled a panel of global dermatology and psychology experts, with the objective to review the current research in the role of psychological comorbidities, focusing on stress and the interplay of the hypothalamic-pituitary-adrenal axis (HPAA) in psoriasis. In addition, the committee also sought to describe the current psychological treatment strategies that have been studied in psoriatic patients with recommendations for clinical practice. The following report synthesizes the presentations and panel discussions from this meeting.Andrea Evers discussed the connection between stress reactivity and psoriasis. While the role of stress in the escalation of skin disease and psoriasis is a common patient-reported phenomenon, there have been relatively few experimental studies on the correlation between psoriasis and stress exposure . A questionnaire study of dermatologists in The Netherlands demonstrated that on average, dermatologists referred only eight patients per year to a psychologist or other psychosocial professional and 50% of dermatologists have never referred to a psychologist or other psychosocial therapist , including one-on-one, group-based, and online programs, is probably the most widely studied treatment with clear evidence that an emotion-focused CBT approach can have a positive effect on psoriasis activity, distress, and quality of life, especially if therapy is tailored to the individual.  is a non-profit charity organization that received unrestricted sponsorship from Abbvie & Amgen to support the workshop. The sponsors had no influence on the content of the program or the viewpoints in this manuscript.Alexandra B. Kimball is an Investigator and Consultant and receives grants and honoraria from Abbott, Centocor and Amgen. Julia Schwartz has partial fellowship funding from Janssen.


Axonal connections between the left and right sides of the brain are crucial for bilateral integration of lateralized sensory, motor, and associative functions. Throughout vertebrate species, forebrain commissures share a conserved developmental plan, a similar position relative to each other within the brain and similar patterns of connectivity. However, major events in the evolution of the vertebrate brain, such as the expansion of the telencephalon in tetrapods and the origin of the six-layered isocortex in mammals, resulted in the emergence and diversification of new commissural routes. These new interhemispheric connections include the pallial commissure, which appeared in the ancestors of tetrapods and connects the left and right sides of the medial pallium , and the corpus callosum, which is exclusive to eutherian  mammals and connects both isocortical hemispheres. A comparative analysis of commissural systems in vertebrates reveals that the emergence of new commissural routes may have involved co-option of developmental mechanisms and anatomical substrates of preexistent commissural pathways. One of the embryonic regions of interest for studying these processes is the commissural plate, a portion of the early telencephalic midline that provides molecular specification and a cellular scaffold for the development of commissural axons. Further investigations into these embryonic processes in carefully selected species will provide insights not only into the mechanisms driving commissural evolution, but also regarding more general biological problems such as the role of developmental plasticity in evolutionary change. In animals with bilateral symmetry, integration between the left and right sides of the body is crucial for processing lateralized sensory-motor functions. This is accomplished by axonal connections between the two sides of the nervous system, known as commissures. Commissural systems are present throughout vertebrate and invertebrate species  mammals, illustrate natural examples of diversity in the developmental mechanisms involved in commissure formation.Development of commissures entails a sequence of events involving morphogenic area patterning, cell-type specification, neuron-glia interactions, production and reception of guidance cues, axonal growth and navigation, and activity-dependent establishment of contralateral connections. In humans, disorders affecting these events at any stage can prevent the normal formation of the commissures, resulting in mild to severe sensory-motor and cognitive conditions , respectively. At the rostral tip of the prosencephalon, fibroblast growth factor (Fgf) proteins are expressed in a region known as the anterior neural ridge, which then becomes the commissural plate, a structure through which the telencephalic commissures cross the midline Figure . Fgfs ary Figure . This gey Figure , and they Figure . Notablyy Figure , but alsy Figure . Similary Figure . Thus, ten route to their central targets, without reciprocally connecting bilateral regions, the optic chiasm is not considered a proper commissure. Along the roof of the midline, immediately rostral to the posterior commissure, lies the habenular commissure  that terminate contralaterally in pallial, subpallial and diencephalic targets , cross the midline at more dorsal territories within the anterior commissure than axons from the olfactory pallium , which decussate more ventrally within the anterior commissure (Northcutt, At the rostral-most extent of the midline lies the anterior commissure, which in agnathans connect mostly the olfactory bulbs and septum with their contralateral homotopic structures, as well as with hypothalamic targets (Nieuwenhuys and Nicholson, lamina terminalis (see Figures A crucial milestone in vertebrate evolution that resulted in several behavioral and anatomical adaptations, including a significant increase in brain complexity, was the colonization of terrestrial niches by the ancestors of modern tetrapods. In particular, the telencephalic pallium underwent considerable increase in size and number of connections, acquiring further complexity in mammals with the evolution of the six-layered isocortex. Consequently, the telencephalon of tetrapods evolved additional commissures that provide interhemispheric connections between pallial regions. Early neuroanatomists described a distinct commissure in the telencephalon of reptiles, termed the pallial commissure (cpal; Figure Sensory adaptations may also have influenced the evolution and diversification of telencephalic connections, including commissural systems. Colonization of land involved the evolution of aerial respiration and the emergence of an accessory olfactory system specialized in pheromone detection (for a review, see Suárez et al., cornu ammonis between hemispheres, as well as heterotopic fibers connecting the hippocampus with the entorhinal cortex (Steward, a structure of brain which is intermediate of that between placental Mammalia and Birds” (Owen, fasciculus aberrans, that joins the dorsal aspect of the anterior commissure through the internal capsule (Figure Similar connectivity patterns are found in amniotes, such as reptiles and birds, where the anterior commissure connects mostly subpallial and olfactory-recipient regions from both hemispheres (Zeier and Karten, Steward, . The evos” Owen, ; p. 92. e Figure . Again, e Figure .fasciculus aberrans and finally the hippocampal commissure, whereas in placental mammals the anterior commissure forms first, followed by the hippocampal commissure and then the corpus callosum (Ashwell et al., The events that led to the evolution of the mammalian isocortex in general, and eutherian corpus callosum in particular, cannot be fully understood from the fossil record and therefore require comparative developmental and molecular approaches. However, fossil skull endocasts of early ancestors of modern mammals suggest that the primitive mammalian brain was dominated by olfactory structures, including a large piriform cortex, and a small isocortex (Rowe et al., lamina terminalis, whereby providing a substrate for convergence and decussation of commissural axons (Figures Shh enhancer, and can also regulate the expression of Fgf8 prior to telencephalic midline formation (Lagutin et al., As discussed previously, patterning of the telencephalic midline in mouse embryos, including the establishment of dorso-ventral territories of commissure formation, is directed by the spatially defined expression of a conserved set of morphogens. The medial pallium/cortical hem expresses Wnt/BMPs, the basal prechordal plate expresses Shh, and the anterior neural ridge, or presumptive commissural plate, expresses Fgfs Figure . These m Figures . From do Figures . The for Figures . In cont Figures . This reCollectively, these genetic patterning studies suggest that initial formation of the commissural plate involves the morphogenic activity of BMP/Wnt and Shh to establish pallial and subpallial territories, respectively, and that the subpallium is then further refined into septal and preoptic regions through Fgf8 signaling. Thus, the specific location through which commissural axons cross the midline depends on the early molecular patterning of the commissural plate, whereby pioneer axons of the corpus callosum cross through the same pallial domain of the dorsal hippocampal commissure, while the ventral hippocampal and anterior commissures form at the septal and preoptic domains, respectively Figure . Taken tAnother important aspect of commissure development that could also account for evolutionary events that led to commissure diversification involves axon guidance and targeting. Following the induction and patterning of the telencephalic midline, growing commissural axons are channeled toward and across the midline by a number of glial cell populations present throughout mammal species (Silver et al., After crossing the midline, callosal axons grow into the contralateral hemisphere and innervate homotopic (Yorke and Caviness, In the context of evolution and development of forebrain commissures, a number of brain features can be distinguished as highly conserved throughout vertebrates, the first being a requirement for interhemispheric communication of the two halves of the CNS. The presence of commissural systems throughout bilaterians reflects a computational requirement of interhemispheric coordination for normal behavior. Second, the conservation in vertebrates of a defined set morphogen expression at the telencephalic midline indicates an important developmental event that directs both the identity patterning of brain areas and wiring of commissural axons. Third, another feature of commissural systems shared by vertebrates is the co-occurrence of decussating fibers that project to heterotopic regions with commissural fibers connecting homotopic regions between hemispheres. Moreover, the presence of profuse heterotopic projections in forebrain commissural pathways of early-branched vertebrates suggests that homotopic projections arose as a refinement of the former kind. Finally, a topographical arrangement of axons within the commissural tracts according the place of origin of their cell bodies can also be recognized as a general feature of commissural systems. Moreover, the origin of new commissures, such as the pallial commissure in early tetrapods and the corpus callosum in eutherian mammals, seems to involve the rerouting of a specific population of topographically arranged axons through preexistent commissural substrates. Such examples of axonal rearrangement can be found in congenital cases of callosal malformations in humans (Tovar-Moll et al., Although there is currently little evidence to allow speculation about the precise mechanisms that led to the evolution of the corpus callosum in eutherian mammals, an evolutionary developmental approach integrating current gene manipulation techniques in carefully selected animal models may shed light on this fascinating topic.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hematopoietic stem cell transplantation (HSCT) is a definite cure for many hematological diseases. With the increasing indications for HSCT and its relatively low cost in Indian subcontinent, an increasing number of patients are opting for this procedure. We retrospectively analyzed the cost of one hundred sixty two HSCTs done at our center in the last three years. The median cost of autologous transplant was USD, $ 12,500  and the median cost of allogeneic transplant was $ 17,914 . The cost of HSCT is cheaper here compared to that in developed countries and success rates are nearly equivalent. The major factors contributing to the cost are related to the complications post-transplant mainly infections and graft versus host disease, which are also the reasons for the increased stay in the hospital. One fifth of the world population resides in India. The burden of hematological diseases both malignant and non-malignant is huge in the country. About 10,000 children are born with thalassemia major each year, and about 6,000 cases are diagnosed with aplastic anemia per year.Between January 2011 and September 2013, a total of 162 patients with hematological diseases received HSCT at the Bone marrow transplant (BMT) center, BLK Superspeciality Hospital, New Delhi. The study included patients with thalassemia major, leukemia, lymphoma, aplastic anemia, multiple myeloma and others. Written informed consent for HSCT was provided by patients after a discussion of the risks and benefits of each method with the patient. The total cost included the cost of chemotherapy, stem cell/bone marrow harvest, antibiotic usage, supportive care with blood, platelet transfusion and growth factors, the hospital stay charges, the investigation charges and consultation fees. The data was obtained from computerized hospital information system. All patients were treated in Hepa-filtered BMT rooms in the 12 bedded BMT unit. Patients who expired before engraftment were excluded. The cost of outpatient follow-up or subsequent admissions was also excluded. The study also excluded the cost of procurement of matched unrelated donor harvest charges and the cost of HLA typing and donor assessment. Peripheral blood stem cell harvest was done in the blood bank by trained apheresis team; bone marrow harvest was done in the operation theater under general anesthesia. Transplant program employed a primary transplant team which conducted and monitored all pre-transplantation and post-transplantation care, supported by medical and pediatric intensivists. The study was approved by the Institutional Review Board and hospital’s Ethical committee.Conditioning before HSCT consisted of high-dose chemotherapy or reduced conditioning regimens with or without antithymocyte globulin. The commonly used regimens were busulfan/cyclophosphamide, fludarabine/cyclophosphamide/antithymocyte globulin, fludarabine/melphalan, thiotepa/triosulphan/fludarabine, melphalan and carmustine/etoposide/cytarabine/melphalan . ConditiA total of 162 consecutive patients were evaluated for the cost of the procedure, focusing on the inpatient costs, till discharge from the hospital . The medHSCT is the cure for many hematological and non-hematological diseases; and in developing countries, where socio-economic status is a major limiting factor, the cost factor associated with BMT is an important issue. We retrospectively analyzed the costs of bone marrow transplantation in 162 consecutive patients transplanted in a tertiary care centre in northern India. The period of cost calculation was from the day of admission to the hospital for transplantation to the day of discharge. The median stay was 33 days (range 17–56 days). The median cost of autologous transplant was $ 12,500  and the median cost of allogeneic transplant was $ 17,914 . The major cost was of the drugs  and the blood products.We also compared the cost of bone marrow transplants (BMT) done at our center with the cost of BMT in developed countries. Because of wide variations in the conditioning protocols and GVHD prophylaxis used, differences in supportive care practices, physician’s discretion in using the available resources and the different time periods of treatment follow-up, included in various studies, the cost factors are difficult to be compared. Still, when compared to the cost of the transplant in Europe and USA where it ranges from $30,000 to $88,000 for a single autologous transplantation to $200,000 or more for a matched unrelated myeloablative allogeneic procedure,Cost of transplant also varies with the type of transplant , source of graft (sibling or matched unrelated), intensity of conditioning regimens used etc.The variation in the cost of the transplant is also directly related to the complications post transplant. These are a) infections , b) requirement of blood product transfusions, particularly because of the delay in platelet engraftment- requiring irradiated single donor platelets, c) Intensive care- patients requiring admission in medical intensive care units or ventilator support, d) onset of severe acute GvHD. Also, various infective and non-infective complications can develop later on, following the discharge from the hospital, and can increase the total cost of stem cell transplantation.15In developing countries, the advantage of opting for transplant in patients with thalassemia major seems beneficial and much more cost effective than lifelong transfusion, chelation and investigation cost,The weakness of our study is that we have analyzed the cost of transplantation from the period of admission for transplantation till discharge after engraftment. The cost of successive admissions and the cost of GvHD prophylaxis and anti-microbial prophylaxis and the cost of regular out-patient follow-up and investigations were not included in the study. Moreover, the cost effectiveness was also not analyzed.The cost of bone marrow transplant till engraftment is much lesser in developing countries compared to developed countries with nearly equal success rate. In spite of this, many patients are not able to afford a much needed life saving procedure because of poor financial support. Further interventions to reduce the cost of the transplant to make it more affordable to the general population needs to be searched, considering the growing burden of patients with hematological diseases.


The inability to diagnose numerous diseases rapidly is a significant cause of the disparity of deaths resulting from both communicable and non-communicable diseases in the developing world in comparison to the developed world. Existing diagnostic instrumentation usually requires sophisticated infrastructure, stable electrical power, expensive reagents, long assay times, and highly trained personnel which is not often available in limited resource settings. This review will critically survey and analyse the current lateral flow-based point-of-care (POC) technologies, which have made a major impact on diagnostic testing in developing countries over the last 50 years. The future of POC technologies including the applications of microfluidics, which allows miniaturisation and integration of complex functions that facilitate their usage in limited resource settings, is discussed The advantages offered by such systems, including low cost, ruggedness and the capacity to generate accurate and reliable results rapidly, are well suited to the clinical and social settings of the developing world. The long-term social and economic stability of a country is highly dependent on improved health. The worldwide advancement in health and technology in the last 50 years is far greater than in the previous 500 years . In spitPOC testing is a necessary component of affordable worldwide healthcare allowing rapid testing at, or near, the site of patient care. In limited-resource or non-existent healthcare settings, or where it is very hard to physically access relevant facilities, POC approaches can save hundreds of thousands of lives every year . The gloetc., is increasing at a much higher rate in developing countries than in the developed countries. Every year, acute respiratory infections account for approximately 4.3 million deaths, 1 million people die from malaria, and about 5 million from AIDS and tuberculosis in the developing world [At present, the global population is more than 7 billion with a growth rate of around 1.14% per year, and it is expected to reach 9 billion or above by 2050. Developing countries account for 84% of the global population, with Asia accounting for approximately 60%, contributed to mainly by the two most populous countries, China and India [ng world . In 2013ng world . DevelopAccess to diagnostic tests and quality community screening can dramatically improve public health in the developing world. Accurate diagnosis plays a crucial role in identifying the incidence and root cause of diseases at both the individual and population levels. Most of the commercially available diagnostic tools for diseases are insufficient in meeting the needs of the limited-resource setting or a weak infrastructure-based healthcare set-up. These shortcomings include long TATs for testing with associated delay in availability of data resulting in late decision making. The development of point-of-care (POC) tests for limited-resource settings is an active and promising area for improving disease management. In addition to the increase in private-sector investment in diagnostics development, a number of initiatives funded by the public sector, including the European Union’s Seventh Framework Programme (FP7) and Horizon 2020, the UK’s Department for International Development, the Foundation for Innovative New Diagnostics, the U.S. Global Health Initiative, and the Bill & Melinda Gates Foundation are currently running with the objective to develop new diagnostic tools to support the healthcare systems of the developing world. An ideal diagnostic test for a limited-resource setting should have the following characteristics: be user friendly; generate results rapidly, allowing medical personnel to diagnose disease and start immediate treatment; be robust (no requirement for refrigerated storage); avoid requirements for complex pre-processing of biological samples; be portable, inexpensive, and highly sensitive; and have the requisite specificity . LateralLateral flow or Lateral Flow Immunoassay (LFIA) technology is the most simple and successful rapid diagnostic testing platform derived from the latex agglutination test developed by Singer and Plotz in 1956 . Since iThe human pregnancy test was the key application driving the initial development of a rapid test platform; this made great strides in the 1970s in decentralized testing. Initially, the rapid tests for infectious diseases, including tests for human immunodeficiency virus (HIV-1 and HIV-2), TB, and hepatitis B were introduced in a dipstick format in developing countries. In the late 1980s, the first immunochromatographic strip (ICS) lateral flow format was introduced for disease diagnosis. Since then the technology, its applications, and the associated industry have continued to evolve. These test platforms have attractive performance features for developing countries, including stability for more than one year, precise performance, interpretation by minimally trained users, usability with a wide range of specimens, and no refrigeration required during shipping and storage. There are several thousand untrained persons performing rapid testing in developing countries . GloballPlasmodium falciparum malaria testing based on LFIA has also shown promising sensitivity for detection of disease and has benefited the developing countries in terms of reduced mortality [The development of LFIA tests for use in developing countries has focused mainly on infectious diseases, including HIV, Strep A/B, legionella, malaria, respiratory viruses, influenza, meningitis, filariasis, tuberculosis, measles, mumps, rubella, chlamydia, and gonorrhea . The marortality ,21.LFIA platforms have also impacted the cardiac disease diagnosis segment. The rapid results using LFIA platforms in cardiac ischaemia have resulted in the rapid diagnosis and precise medical treatment given at right time ensuring reinstatement of the blood flow to the heart. Using LFIA-combined with cardiac markers ) has improved the early diagnosis of ischaemia resulting in better patient management . For exaLateral flow immunoassay (LFIA) is based on the use of a nitrocellulose, polymer, paper, or other composite substrate membrane which facilitates the separation, capture, and detection of the target analyte(s) of interest. The various components of a LFIA have the capacity to transport fluid, including blood or serum, under capillary action, and thus no external pumps are required.There are various formats for lateral flow assays. In a simple capture format  the targIn practice, the system works as follows. Briefly, a sample is introduced on to the sample pad through the sample application window present on the plastic casing, as shown in The different constituents of a lateral flow test and the raw materials used for each constituent that play a vital role in the quality of the results generated are critically assessed in the next sections.The high specificity and selectivity of the biorecognition element are key for the performance of the assay. There are many different recognition elements that can be employed but antibodies are by far the most common and well established. Antibodies used in LFIA can be monoclonal, polyclonal, or both together, but increasingly recombinant antibodies are also being used. Traditionally polyclonal and monoclonal antibodies have been used extensively. Both have their own advantages, including the high specificity of monoclonal antibodies, which helps in decreasing non-specificity and cross-reactivity, while the ability of polyclonal antibodies to recognize multiple epitopes on a single antigen, as well as their low cost production in bulk quantities, are valuable properties. Many rapid tests employ 2–3 types of antibodies, e.g., for binding specifically to the target antigen, for carrying the label for visualisation, and for acting as a control. The stationary phase includes antibodies immobilised at the capture line location  to capture antigen, and another at the control line. The mobile phase contains the antibody bound with a label, e.g., enzymes, fluorescent tags, gold nanoparticles, quantum dots, or latex beads, which first interact with the target analyte in a direct or sandwich immunoassay format and then come in contact with the immobilised capture antibodies. Recently, the potential of using recombinant antibodies is being explored. This derives from the fact that their specificity can be carefully tuned to allow either broad or highly specific binding, allowing the analysis of groups of related targets or analysis of an individual target. They can also be functionalised to allow high levels of immobilisation with optimal orientation. In addition, the labels may be molecularly linked to the antibody, e.g., use of green or other fluorescent proteins, thus avoiding labelling steps which may lead to conjugations that may reduce or even inhibit the antigen- binding capacity of some antibody molecules. Issues related to stability may be overcome by additional engineering to create formats e.g., Fabs or scAbs, that work well in LFIA. In addition to antibodies, aptamers, affimers, more complex molecularly imprinted polymers, and several other binders are being explored as promising alternative capture probes ,26,27. TCellulose, glass fibre, rayon, or cross-linked silica are common materials used to make sample application pads. The major role of the sample pad is to allow continuously uniform migration of the sample to the conjugate pad whilst making the sample compatible with the other components of the test system. This is usually achieved by pre-treatment of the sample by separating the plasma or serum from whole blood and may involve trapping of erythrocytes and other cells, adjustment of pH, and elimination of interferents, in order to allow efficient interaction of the target analyte with antibodies and conjugates.The sample migrates from the sample pad to the conjugate pad where it mixes with the dried conjugates . Conjugate pads are usually made up of glass fibres or polyesters and are often pre-treated with different proteins, polymers, and surfactants to make them hydrophilic in order to ensure stability and optimum release of the conjugate. The material composing a conjugate pad often plays a vital role in determining the overall shelf-life of the LFIA test by ensuring the stability of antibodies, labels, and the associated conjugates, and allowing release of the conjugates from the pad . During A wide variety of membrane types and pore sizes are commercially available. The selection of the membrane depends on the analyte and the application of the test. Typically, a hydrophobic nitrocellulose (high protein binding), glass fibre (non-protein binding), or cellulose acetate (low protein binding) membranes are used for LFIA. Nitrocellulose membranes are the most widely accepted materials that have been efficaciously applied for LFIA tests due to their distinctive characteristics including high affinity for proteins, ease of wetting, lack of interference with assay, relatively low cost, commercial availability of several versions of nitrocellulose, true capillary flow characteristics, and comparative ease of handling. The reaction area is comprised of two lines or zones on the nitrocellulose membrane, known as the test (or capture) and control lines. The capture line consists of specific antigen or antibodies immobilised to capture the target analyte whereas the presence of the control line confirms the proper flow of the liquid or performance of the assay. Several factors including accurate dispensing of biological components and proper blocking and drying play a key role in refining sensitivity of the assay.The absorbent pad is usually made up of high-density cellulose and is placed at the end of the strip or after reaction region. The main role of an absorbent pad is to maintain the flow of reagents over the membrane and to avoid false positives by not allowing back flow of reagents. It is also called a wick or the sink in an LFIA system.In order to facilitate easy handling and to provide strength to the fragile membranes used in the LFIA system, all capillary beds are usually mounted on a backing material. The materials used for providing the requisite rigidity are typically polystyrene or different variants of plastic which are coated with a pressure-sensitive adhesive to hold all the components together in a defined format. The compatibility of proteins with adhesives over a long period of time plays an important role in maintaining the prescribed shelf-life of the product. The backing serves as a structural support to the LFIA system to which several layers  performing different functions are laminated and the entire entity is housed in a single cassette.The choice of a label used for detection normally depends on the application of the LFIA system. Labels such as latex beads, colloidal carbon, colloidal gold, fluorescent tags, enzymes, streptavidin, or gold nanoparticles, are conjugated with an antibody or an antigen depending on the format of the LFIA system ,29,30. SThe majority of the lateral flow assays use visual detection. The incorporation of gold nanoparticles or latex beads as labels allows visual examination of colours at the test and control lines, resulting in qualitative or semi-quantitative analysis. The generation of rapid results in critical or intensive care unit (ICU) patients is of great assistance to doctors to make an instantaneous decision and initiate treatment. The use of colloidal gold, paramagnetic particles, and fluorescent dyes as labels in lateral flow formats has opened up new horizons for LFIA technology by allowing true quantitative testing. The development of quantitative assays in lateral flow formats requires an additional fluorescence or optical strip reader which allows measurement of the intensity produced at the test and control lines of the strip ,32,33. TAs discussed above, LFIA techniques have allowed quick and simple point-of-care diagnosis of a variety of diseases in different environments. However, their simplicity limits their performance and in many cases more complex devices are necessary for accurate diagnosis. LFIA or immunochromatographic (ICS)-based strips have successfully been implemented for the diagnosis of several transferable diseases such as HIV, tuberculosis, and sexually-transmitted diseases (STDs) in low-resource settings. ICS strips rely on capillary forces to control fluid flow. By the nature of sample addition to such devices, there may be some variations in the amount of sample added initially and actually used in the test and this can result in poor precision. In addition, in many test formats pre-treatment of samples becomes mandatory in cases where the sample matrix is not a fluid or where significant interferents are present. In addition to this, restrictions in the limit of detection of these platforms may restrict their use to the determination of analytes that are highly abundant in the sample tested, such as lymphocytes in HIV tests or pathogenic microorganisms in malaria tests. Hence, the results may be only qualitative or semi-quantitative, even when confirmed with a reader. The large coefficient of variation (CV) associated with an LFIA test also limits the test reproducibility and, therefore, its ability to confirm the presence of the disease . VariablOver the last two decades, microfluidic technologies have experienced a significant growth in applications in the field of diagnosis. Microfluidics-based instrumentation has shown promising results in several of the main laboratory techniques, including blood chemistries, immunoassays, nucleic-acid amplification tests, and flow cytometry . MicroflFully automated instruments located in centralised laboratories are capable of providing highly reproducible, quantitative, and sensitive results. Unfortunately, in developing countries, the necessary infrastructure for these modern laboratories, if available, is often only present in the major cities’ hospitals or in regional centres. Rural areas often lack access to the requisite technology and the necessary trained personal to perform the tests and carry out the subsequent diagnoses, especially where the situation is complicated for patients with multiple concomitant infections and malnutrition. When developing a diagnostic device intended for use in resource-poor areas, it is of utmost importance to ensure it conforms to the stringent performance requirements previously listed in earlier sections of this review. Microfluidic-based systems are able to fulfil these requirements by miniaturising and automating complex diagnostic procedures into a portable device .et al. [n = 38) [Mycobacterium tuberculosis has also been reported. Jing et al. [E. coli 0157:H7, Salmonella, etc.) using a microfluidic chip. The chip consists of two microchannels embedded with magnets, where immunomagnetic separation of the bacteria takes place. Detection is achieved by labelling the captured bacteria with bioconjugated quantum dots which fluorescence [et al. [Several academic and industrial groups have explored the potential of microfluidic-based technologies to be used in resource-poor settings in developing countries. Patterned-paper technology  is beinget al.  analysed[n = 38) . The useg et al.  integratrescence . To date® blood analysis system  in addition to the traditional Premarket Approval (PMA) or the approval via the 510(k) pathway in the U.S.A. For diagnostic tests to be CLIA-waived they must be simple and have a minimum risk for generating erroneous results. This means that sample preparation and fluid handling steps must be minimised, ideally by storing reagents on the microfluidic device and automatically releasing them. Chin et al.  summarisl. [etc. .It is a challenge to integrate sample preparation and processing and detection techniques in a self-contained POC system. Non-instrumental, disposable-only tests would be ideal for delivering health care in low-resources settings as they are cheap and simple to perform. Thus, “disposable-only” microfluidic devices are especially suited for developing country diagnostics. Currently, these non-instrument-based tests are found in three different formats: lateral flow, flow-through, or solid phase. Unfortunately, the limitations in the limit of detection inherent to these platforms makes those disposables with an external reader a better alternative in many cases, in particular when the tested analyte is not very abundant in the sample .et al. [The integration of multiple preparative and analytical microfluidic technologies in a single device allows the use of unprocessed samples to run an entire assay at a POC setting. Preconditioning of samples, when needed, is possible thanks to centrifugation, which allows the purification of plasma from blood, or microfluidic technologies such as an on-chip filter trench where blood cells are sedimented and captured ,48. As met al.  developeBeyond the technical challenges, successful implementation of a diagnostic test in remote settings relies on the ability of the user to operate the test. The development of an appropriate interface is of utmost importance in those low-resource areas where healthcare workers are often not professional clinicians. These individuals would be able to perform basic tests, such as ICS strips for pregnancy or malaria, but may encounter difficulty if they need to prepare the samples or handle different reagents . MicroflAll of the steps involved in the diagnostic procedure should be simple and as culturally independent as possible. Reading a basic instruction manual written in non-technical language should enable the user to operate the test without any further “hands-on” training. The training requirements of a variety of FDA-approved microfluidics-based POC tests are shown in et al. [A diagnostic test usually gives information regarding the presence/absence or concentration of the analyte of interest contained in a biological sample . Healthcet al. , who desOne of the strengths of microfluidics is that they are able to produce sensitive results by detecting the signal produced in a very small area using fluorescence or other more recently implemented but ultrasensitive detection methods. Electrochemical detection, in particular, has been widely used for improving the sensitivity and also often the specificity of traditional tests, generating quantitative testing due to the use of portable electrochemical analysers in POC platforms. For example, the i-Stat blood gas analyser see  combines® reagent discs or the ABORhCard, already described in this review.For POC testing to reach its full potential in a low-resource setting, disposables need to be robust, allowing shipping and storage at ambient temperature. Reagents and antibodies for immunoassays must be stored on the disposable element. To avoid degradation, biomolecules would ideally be stored in a lyophilised state in vapour-proof compartments. This would protect their integrity, allowing them to be stored at ambient temperature and also would reduce the weight of disposables, which would contain only desiccated reagents. If the use of liquid reagents is necessary, they should be contained in a sterile and sealed environment to prevent evaporation . Dry reaTo test the efficiency of the test, a calibration check should be run in parallel to the test, ideally including both positive and negative controls. Thus, a microfluidic platform in which both a standard curve and the analyte test can be simultaneously generated on the same disc or chip is particularly attractive .Microfluidic technologies offer significant benefits for implementation in developing areas. Reducing the cost of materials to be used in the disposables is a must be taken into consideration. To minimise manufacturing costs, microfluidic devices can be made of plastic  or paper, both inexpensive materials appropriate for the fabrication of disposable devices . In addiDeveloping countries have a much higher mortality rate from diseases such as HIV, TB, and malaria due to poor medical infrastructure in comparison to the developed countries. In addition to the communicable diseases, non-communicable diseases are also imposing a growing burden upon the resource-limited countries. The development of inexpensive, low-maintenance, reliable, and easy-to-use diagnostic tools can easily reduce the burden and impact of diseases in these environments. Despite the presence of high disease burdens and an ever-growing need, there are relatively few diagnostic tools that have been designed for fulfilling the specific needs and challenges of the developing countries. Currently, the only highly successful point-of-care (POC) testing platform available in the rural areas of developing countries is the lateral flow assay LFIA strip, which has contributed greatly to the identification of diseases without the requirement for any modern laboratory set-up. In spite of the advantages offered by LFIA strips, there are several inherent associated weaknesses, as indicated by clinicians and researchers, such as poor reproducibility and accuracy, and low sensitivity. Advancements in nanotechnology, microfluidics, and device fabrication have opened up new horizons in the field of diagnostic platforms for their successful placement in low-resource laboratory settings. Their intrinsic advantages, such as small size, manipulation of small volumes of liquids, high capability of integration, and rapid reactions offered by microfluidic technology make them promising candidates as POC diagnostic platforms. The ability of microfluidics based-platforms to accomplish all analytical steps such as sample pretreatment, assay, and detection, on a single easy-to-use, accurate and automatic test system makes them an ideal future diagnostic tool to provide real clinical value in low-resource, laboratory-free settings.


The use of increasingly precise, intelligent neurosurgery instruments that allow intraoperative accuracy, visualization, surgical access, has led to mastery of surgical techniques and the transformation of prognosis, the Mayfield three-pin skull clamp was designed to rigidly affix a patient's head to the operating table during craniotomy drilling and delicate microneurosurgery. However, these instruments are not without risk, since several types of complications have been described. We report the case of depressed skull fracture a secondary to the Mayfield three-pin skull clamp in a patient operated for a meningioma of the posterior fossa as shown in this picture CT.


An eight-year-old girl presented with complaints of intermittent discharge from a pit situated in the medial surface of right pinna since early childhood. The discharge was scanty, foul-smelling and mucoid, and occasionally purulent. The frequency had increased in the last year with development of a swelling just below the pit that often got inflamed and caused pain. On examination, an opening was noted just above the lobule leading to a swelling that reached the post-auricular sulcus . An area , but might be intraauricular or post-auricular. However, the clinical and embryologic implications could be different depending on their location and composition. The pinna is formed by the union of six cartilaginous hillocks (of His) derived from the first two branchial arches. The antitragus, antihelix, lobule and lower portion of helix are formed from the second arch, while the rest of the pinna is derived from the first. Pre-auricular and intra-auricular sinuses/cysts are manifestations of faulty union of the two branchial arch derivatives; they are sort of inclusion cysts/tracts and are innocuous. However, clinically identical cysts, sinuses or tracts may be seen in the posterior aspect of pinna and upper neck whose origin cannot be explained by the inclusion or entrapped tissue theory. In truth, they are duplications of the EAC, and are derived from malformations of the first branchial sinus which contributes to the development of the EAC. These may either be lined by epithelium (type I) or formed by both ectodermal and mesodermal components (type II), thereby often draining in the EAC or in the neck, and are in close proximity to the parotid gland and facial nerve. These make them vulnerable during surgical exploration. This particular child coincidentally had both branchial arch and cleft anomaly clinically presenting as pits in close anatomic proximity. It becomes very difficult in such cases to differentiate an inclusion cyst/sinus from a type I first branchial cleft anomaly which is in intimate relation with the membranous EAC. The situation might be misleading, and congenital syndromic disorders and the involvement of vital structures,,5 both of which are known associations with branchial cleft malformations, would be missed if they are erroneously mistaken for the relatively benign and embryologically distinct preauricular and intra-auricular sinuses. Imaging (sinogram) does contribute, but the differentiation truly lies in the knowledge of the regional embryology and per-operative findings.Peri-auricular pits are congenital stigmata commonly encountered by clinicians when they become infected and symptomatic. They might lead to sinuses, fistulae, cysts, or simply end in blind tracts, and are mostly noted in the pre-auricular region