Effective and safe monitoring techniques are needed by U.S. land managers to understand free-roaming horse behavior and habitat use and to aid in making informed management decisions. Global positioning system (GPS) and very high frequency (VHF) radio collars can be used to provide high spatial and temporal resolution information for detecting free-roaming horse movement. GPS and VHF collars are a common tool used in wildlife management, but have rarely been used for free-roaming horse research and monitoring in the United States. The purpose of this study was to evaluate the design, safety, and detachment device on GPS/VHF collars used to collect free-roaming horse location and movement data. Between 2009 and 2010, 28 domestic and feral horses were marked with commercial and custom designed VHF/GPS collars. Individual horses were evaluated for damage caused by the collar placement, and following initial observations, collar design was modified to reduce the potential for injury. After collar modifications, which included the addition of collar length adjustments to both sides of the collar allowing for better alignment of collar and neck shapes, adding foam padding to the custom collars to replicate the commercial collar foam padding, and repositioning the detachment device to reduce wear along the jowl, we observed little to no evidence of collar wear on horses. Neither custom-built nor commercial collars caused injury to study horses, however, most of the custom-built collars failed to collect data. During the evaluation of collar detachment devices, we had an 89% success rate of collar devices detaching correctly. This study showed that free-roaming horses can be safely marked with GPS and/or VHF collars with minimal risk of injury, and that these collars can be a useful tool for monitoring horses without creating a risk to horse health and wellness. Equus caballus) management is a complex issue incorporating social, economic, emotional, political, and environmental factors. The complexity associated with developing appropriate free-roaming horse management practices has highlighted the need for an improved understanding of free-roaming horse behavior, habitat use, resource impacts, and movement patterns across the landscape. Global positioning system (GPS) collars offer a robust tool to track horse activity with high spatial and temporal resolution. In 1994, Lotek Engineering introduced the first animal-based GPS tracking device Free-roaming horse  and GPS collar technologies are commonly used by wildlife managers and researchers to track and monitor a diversity of wildlife species, ranging from migratory birds Equids, however, those that have incorporated this technology have successfully generated data for the study of animal movement, behavior, and habitat use. For example, Siniff et al. Equus ferus przewalskii) and seven Asiatic wild asses (Equus hemionus onager) in the Mongolian Gobi. In the Australian outback, Hampson et al. Equus burchelli antiquorum) to test the influence of collar weight on animal behavior and location error In contrast, few studies report the use of VHF or GPS collar technology in the study of wild Despite the technology's widespread application, in the United States collars have been barred from use on free-roaming horses that are under the jurisdiction of the Bureau of Land Management (BLM) because of previous incidents of collar-related injuries to horses fitted with VHF collars. The BLM is responsible for management of the majority of free-roaming horses in the United States. Problems included ill-fitting collars and, more importantly, a lack of collar removal that resulted in horses wearing collars for extended periods causing significant injury . Horse necks increase in width from the base of the head (lower mandible/axis) to the point of neck attachment above the shoulders and withers. Collars that are attached too low on the neck towards the withers have high potential for significant movement and associated injury. This is primarily due to slippage that occurs as collars slide up and down the neck while feeding, walking, and running. This rubbing motion can create hair wear and if excessive, open wounds and sores.In the United States, free-roaming horse welfare and safety is a paramount management issue and because of safety concerns, there has been a limited use of VHF/GPS collar technology for improving our understanding of free-roaming horse ecology and management. The purpose of this study was to evaluate VHF/GPS collars and assess their potential to cause injury to free-roaming horses. This includes an assessment of collar design, degree of injury, and collar detachment reliability. Note that the terms free-roaming, free-ranging, feral, and wild horse are used interchangeable by horse management agencies and in scientific literature, however, these terms all refer to horses that live in an untamed state but have ancestors that were once domesticated. In this paper, we refer to these animals as free-roaming horses.We used two separate study locations to evaluate the utility and safety of VHF/GPS collars in characterizing free-roaming horse movement and habitat use patterns: the Roaring Springs Ranch (RSR) in southeast Oregon and the Sheldon National Wildlife Refuge (SNWR) in northwest Nevada . The RSRArtemisia tridentata Nutt. ssp. vaseyana (Rydb.) Beetle), bottlebrush squirreltail (Elymus elymoides (Raf.) Swezey), Idaho fescue (Festuca idahoensis Elmer), bluebunch wheatgrass (Pseudoroegneria spicata (Pursh) A. Love), and Sandberg's bluegrass (Poa secunda J. Presl).The RSR is located on the western side of Steens Mountain (42.36108° N 118.78923° W) in the northernmost extent of the Great Basin, High Desert Ecological Province A. tridentata ssp. vaseyana and ssp. wyomingensis Beetle and Young), little sagebrush (A. arbuscula Nutt.), antelope bitterbrush (Purshia tridentata (Pursh) DC.), bottlebrush squirreltail, Idaho fescue, and bluebunch wheatgrass. Several tree species such as quaking aspen (Populus tremuloides Michx), western juniper , and curl-leaf mountain mahogany (Cercocarpus ledifolius Nutt.) are found in scattered stands throughout the SNWR.The SNWR is located along the southern Oregon border to the north and near the California border to the west . The SNWR covers approximately 232,694 hectares of sagebrush-steppe habitat with elevations that range between 1,200–2,100 m. Average annual precipitation ranges between 18 and 33 cm. Plant communities in this region are dominated by big sagebrush  and eight custom collars VHF/GPS collars to free-roaming horses within the Sheldon National Wildlife Refuge in August 2009 . Horses All collars were deployed on adult mares ranging in age from four to 24 years old and ten of the mares were attended by foals at the time of collaring. During the initial collar deployment in August 2009, all collars were outfitted with both a breakaway device  and a commercially available remote timed-release device (Lotek mini drop off) set to detach with a delay of approximately 53 weeks .Following the initial deployment in August 2009, all of the breakaway devices installed on the six commercial collars activated prior to the pre-programmed date for the timed-release devices. The commercial collars were retrieved from the field, and were refurbished by the manufacturer for re-deployment in October 2010 on SNWR. Because the timed-release devices were still intact and operating when retrieved, they were not replaced during refurbishment and instead reset in the field according to the manufacturer's instructions. The refurbished commercial collars deployed in October 2010 were outfitted with only the reset timed-release detachment devices.This study was carried out in strict accordance with the protocol approved by the Brigham Young University Institutional Animal Care and Use Committee . No permits were required to perform this research. The pilot study was performed on private land under the authority of the landowner while the field study was performed with permission and guidance of the USFWS on the Sheldon National Wildlife Refuge. Our research did not involve Federally- or State-listed endangered species or species of concern. The owners of the domestic horses granted permission for their animals to be use in this study.Hair wear includes thinning and breakage of hair where friction is enough to provide visible evidence of contact with the collar. In approximately 30% of the cases, hair wear at the RSR pilot study was sufficient enough to expose the skin, however, as this took place over an extended period, the underlying skin adapted by thickening and strengthening the epidermal structure and the skin remained pliable with no evidence of callus formation or loss of elasticity. In no case was the skin integrity compromised. Collars were placed high on the neck to reduce collar movement while also limiting the potential for collar damage associated with horse to horse interactions , or getting the collar caught in vegetation or on fences. Moreover, collaring only mature animals prevented the possibility of an immature animal outgrowing the collar and causing injury. During the pilot study, one of our main goals was to evaluate study horses for potential issues or injury attributed to the collar. The collars were installed behind the head and ears  and initObservations over the first 4 weeks following deployment included: 1) slight to moderate abrasions or callus on the jowl at the jugular furrow; and 2) slight to moderate wearing in the hair at the widest point of the neck and on the mane. We also found that the remote collar detachment device was causing skin irritation by rubbing along the jowl . As a reDuring the pilot study, two of the breakaway devices detached before 40 days while the remaining three collars with breakaway detachments were intact when the study ended and the collars removed in August 2009. Six of the seven (87%) remote-detonated collar detachment devices detached as expected after 93 days deployed. The reason for the single failure is unknown.During the pilot study, we documented that several of the domestic geldings were able to slip the upper portion of the collar band over one or both ears; we did not see this occur with any of the mares. The exact reason for this is unknown, but to reduce the potential for harm to free-roaming male horses, the decision was made to only collar mares during the in-field portion of the study. In addition, we suspected that intrasexual fighting among male horses would likely result in substantial collar damage For the custom-built collars deployed in August 2009, the VHF malfunctioned almost immediately and stopped transmitting from seven of the eight collars making the collars irretrievable from the field. The remaining collar was successfully retrieved field via a functioning VHF transmitter and a second collar was found by chance and recovered by a member of the public. All eight horses who received custom-built collars were recaptured during subsequent helicopter gathers between 2010 and 2012. None of these individuals were wearing a collar when recaptured and under examination showed no evidence of lasting injury as a result of the collar . Because the collars were irretrievable from the field due to the VHF malfunction, it is unknown exactly when and which collar detachment device activated first (the breakaway or timed-release), but the end result was the same with all collars detaching successfully.The VHF transmitters in the commercial collars transmitted a signal throughout the study. For the commercial collars deployed in August 2009, all six had their breakaway device detach before the set remote timed-release date of approximately 370 days since deployment, ranging from 122 to 254 days . The comCollar wear during the in-field study was as expected from that observed following the collar modifications during the pilot study. During the course of the in-field study, we located the bands with marked individuals via VHF by air or ground every 1 to 2 months, and all of the mares appeared from a distance to be in good health and wearing the collars without issue. These observations included individuals wearing collars that had stopped transmitting a VHF signal, but were able to be located due to their association with mares whose collars were transmitting correctly. Fourteen mares were incidentally captured during subsequent gather efforts and under direct examination showed very little to no evidence of effects related to the collar regardless of type of collar deployed. If evidence of the collar was present, we observed only a small callus or a slight wearing of the hair along the neck and mane. Of the two individuals whose timed-release detachment device did not function properly, one was recaptured in 2013 still wearing the collar and showed no injury or damage as a result of the collar even after 3 years of continuous wear .Ill-fitting collars and problems associated with them clearly influence research results and have implications for ethics within the wildlife profession We tested the custom-built collars due to the greater cost savings however, we experienced issues with their functionality, likely due to inexperience with the construction and potentially rougher handling by horses than other species. We observed several instances where a commercial collar took a direct kick from another horse and continued to function normally. For those custom-built collars we could recover, in several locations soldered joints in the electronics were broken leading us to suspect there were flaws in the construction. Collars built following the Clark et al. In evaluating the collar detachment devices, the collars detached successfully in 89% of the cases. We suspect issues related to failure in the second deployment of the timed-release devices were related to our attempts to field-reset them, where two detached at the expected time, two detached at twice the expected time, and two failed to detach. For those collars that were not removed by either a remote-detonated or a timed-release device, the fail-safe breakaway devices were 100% effective. The downside of the breakaway devices is a lack of control over when the collar will detach, which in our environment ranged roughly between 4 and 8 months. This timing would be expected to be variable depending on the thickness of the rubber tubing used, climate  of the study area, and time of year (winter vs. summer). We expect that thicker tubing, would last longer, however, tubing thicker than the diameter tested in this study would be difficult to tie into the collar and may subsequently falter.The potential exists for horses to be injured when collars get caught in trees or fencing. Although we did not specifically monitor for these events, we saw no evidence of any injuries that could be attributed to horses being trapped in fencing or trees because of collars. Moreover, breakaway devices may provide protection against this type of injury, however more research specifically on the strength of the rubber tubing is required to fully address this question.Based on our experience in this study, collar fit is an important consideration when using VHF/GPS collar on free-roaming horses. By maintaining a natural oval shape and fitting the collar high on the neck, collar movement and subsequent wear was significantly reduced. In addition, collar detachment devices should be installed along the collar in locations to ensure they do not cause excessive wear along the horse's jowl.Equid studies, none reported observations of significant collar-related injuries to study animals Injury to study animals can occur when using GPS/VHF collars. Krausman et al. Although not reported in this paper, the data collected by our GPS collars provided useful information with respect to the effectiveness of geospatial data in evaluating free-roaming horse ecology including landscape-level movement patterns The use of VHF/GPS collar technology is critical to understanding how free-roaming horses, particularly in the Western United States, move across the larger landscape and use increasingly scarce resources. Lack of this information has contributed to the management complexity of this species


Proteolytic enzymes  are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences. Proteolytic enzymes are capable of hydrolyzing peptide bonds and are also referred to as peptidases, proteases or proteinases .The physiological function of proteases is necessary for all living organisms, from viruses to humans, and proteolytic enzymes can be classified based on their origin: microbial , plant, animal and human enzymes can be distinguished.N- or C-terminal ends of the substrate. Aminopeptidases (Proteolytic enzymes belong to the hydrolase class of enzymes (EC 3) and are grouped into the subclass of the peptide hydrolases or peptidases (EC 3.4). Depending on the site of enzyme action the proteases can also be subdivided into exopeptidases or endopeptidases. Exopeptidases catalyze the hydrolysis of the peptide bonds near the ptidases  can libeptidases , while pptidases  cleave pBased on the catalytic mechanism and the presence of amino acid residue(s) at the active site the proteases can be grouped as aspartic proteases, cysteine proteases, glutamic proteases, metalloproteases, asparagine proteases, serine proteases, threonine proteases, and proteases with mixed or unknown catalytic mechanism .The current classification system further classifies the proteases into families based on sequence similarities, furthermore, homologous families are grouped into clans using a structure-based classification ,4. ClassAction of the proteolytic enzymes is essential in several physiological processes, e.g., in digestion of food proteins, protein turnover, cell division, blood-clotting cascade, signal transduction, processing of polypeptide hormones, apoptosis and the life-cycle of several disease-causing organisms including the replication of retroviruses ,6. Due tIt was estimated previously that about 2% of the human genes encode proteolytic enzymes  and due Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require the use of them, including the production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, use of proteases in cell culture experiments and in tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing, and proteolytic digestion of proteins in proteomics.This review focuses on the application of proteolytic enzymes in life sciences, especially in the field of molecular biology. The summary table of proteases discussed in this review  containsE. coli DNA polymerase I enzyme. While the holoenzyme has 5'→3' polymerase, 3'→5' and 5'→3' exonuclease activities, the Klenow fragment has only the polymerase and the 3'→5' exonuclease activities. The Klenow fragment has several applications in the recombinant DNA technology, including the labeling, sequencing, and site-specific mutagenesis of DNA.The Klenow fragment is the large fragment of the The enzymatic method to release the large protein fragment from the DNA polymerase I holoenzyme by proteolysis was published in 1970 . SubtiliE. coli strains which carry the gene of large fragment of DNA polymerase I, therefore, the proteolytic production of Klenow fragment has mainly historical significance.Nowadays, commercially available Klenow fragment is produced in recombinant ways in While enzymatic peptide synthesis has been frequently used to synthesize peptides for pharmaceutical and nutritional purposes , this med-Leu-l-Leu-NH2 synthesis [d-Leu that is activated by carbamoylmethyl (Cam) ester and chymotrypsin (E) form the enzyme-substrate complex first and after that the covalently linked Z-d-Leu-E intermediate with the loss of the carbamoylmethyl ester. If this intermediate is attacked by water, hydrolysis occurs, which results in the Z-d-Leu-OH fragment. However if a more powerful nucleophile  is present in the media, the enzyme produces a peptide bond instead of the cleavage [d-Leu-l-Leu-NH2 dipeptide may be formed in the presence of H-l-Leu-NH2 nucleophile app and the rate at which the peptide product is hydrolyzed. The (KT/KH)app values of proteases used for kinetically-controlled synthesis are in the range of 102–104 [The acyl donor activating agent must not only be an ester, but an amide or a nitrile as well. Only serine or cysteine proteases can be used to perform the kinetically controlled peptide synthesis, as these enzymes can act as transferase and hence are able to catalyze the transfer of an acyl group from the acyl donor to the nucleophile through the formation of a covalent acyl enzyme intermediate. Papain, thermolysin, trypsin and α-chymotrypsin are mostly used for kinetically-controlled peptide synthesis . The yie 102–104 .In the case of the equilibrium-controlled synthesis the process is the reverse of hydrolysis. Important problems of this enzymatic method of peptide synthesis are the low reaction rates and the need of increased yield because proteases do not alter the equilibrium of the reaction. A high amount of the enzyme is often required together with the precise reaction conditions to drive the equilibrium towards synthesis . A highe(a) Precipitation of the product is the classical method. When certain soluble carboxyl amine components are used as reactants the products will precipitate and the reduced concentration of soluble products will drive the equilibrium towards synthesis. In this case the yield of the product depends on the concentration of the starting materials and can be determined by the solubility of the product .(b) In a biphasic system the enzyme acts in an aqueous environment that is surrounded by a water immiscible medium; the water content of the system is around 2–5%. The reactants (dissolved in high concentrations) can diffuse from the organic phase into the water until the equilibrium is reached; the enzyme-catalyzed synthesis is followed by the diffusion of the products back into the organic phase. The organic phase reduces the dielectric constant of the medium and thus the acidity of the carboxyl group of the acyl donor as well, which in turn promotes the synthesis of the peptide bond instead of hydrolysis . This me2 is more efficient in hydrophobic organic solvents; adding water in sub-saturating concentration increases the yield of the chymotrypsin-catalyzed peptide synthesis [(c) The dissolved state system can be used for the synthesis of water-soluble products . In this environment forcing the reaction towards peptide synthesis requires the mass action, the addition of a water-miscible organic co-solvent in high concentration or the excess of one reactant. In serine protease-catalyzed reaction in water, the rate determining feature is the acylation of the enzyme while the product yield at equilibrium depends on the partition coefficient and the ratio of the aqueous and organic volumes . The chyynthesis .use of enzymes in organic solvents have several advantages compared to aqueous solvents which have led to their widespread application: the thermodynamic equilibrium can be shifted towards synthesis, the undesirable side reactions can be reduced, the nonpolar substrates are more soluble in organic solvents, the separation process and enzyme recovery is more effective in a low water-containing environment [The ironment ,27,28,29ironment ,29 can mmodification of biocatalysts by protein engineering [However, the use of enzymes in organic solvent has also disadvantages such as the unfavorable effects of the organic solvents on enzyme activity and stability. The ineering ,31 and/oineering  for peptSubtilisin has been extensively studied and engineered via site-directed mutagenesis to make it more capable of peptide bond formation in aqueous solution . Single Pseudomonas aeruginosa. Considering the high structural similarity of these enzymes, the synthetic activity of PST-01 protease was increased by the Y114F mutation [Thermolysin has a higher synthesis rate compared to the solvent stable PST-01 protease from mutation , moreoveChemical modification is also an efficient method to modify the properties of enzymes used for peptide synthesis. Thiol-subtilisin, in which the serine residue has been chemically changed to cysteine at the active site, shows an enhanced aminolysis to hydrolysis ratio in aqueous solution and in dimethyl sulphoxide. The stability of proteases can also be increased by chemical modification e.g., a hydrophilic carbohydrate-polyacrylate polymer coat can make the enzymes highly active and stable in polar solvents and more resistant against thermal inactivation .Immobilization of proteases is the most frequently applied method for the recovery of products without great loss of the catalysts, which greatly decreases the cost of the synthesis. This approach also ensures better operational stability of biocatalysts and control of the reaction. Enzyme immobilization techniques can be divided into five groups: (a) covalent attachment to solid support; (b) absorption on solid support; (c) entrapment in polymeric gel; (d) crosslinking with bifunctional reagents and (e) encapsulation ,34.Substrate engineering means the manipulation of the leaving group and is a powerful tool to increase the specificity of the proper enzyme and/or increase the rate and the yield of the reaction [reaction .Protease-catalyzed synthesis of stereochemically modified peptides is also a preferable application compared to chemical synthesis due to stereospecificity of the proteases.Proteases can bind not only natural substrates, but also specifically designed substrate mimetics, which are also very useful tools to increase the yield of peptide synthesis. Substrate mimetics can bind to the active site of the enzyme and in this way proteases can be used for the synthesis of products containing non-specific amino acids. The undesired cleavage of the newly synthesized peptide bonds can be avoided using this method and it is not required to change the properties of the medium or the enzyme .The production of peptides with amides at their C-termini is a great challenge for enzymatic peptide synthesis, but amidation may be required to retain biological activity.Generally, the first step of nucleic acid isolation protocols is the lysis of the biological material containing the DNA or RNA of interest. Before the purification and concentration of nucleic acids the contaminating proteins and other macromolecules have to be removed from the sample. Undamaged nucleic acids can be isolated when the degradation of the DNA and RNA present in the sample is avoided by the inhibition and removal of DNases and RNases. The nucleases can be inhibited by the addition of chelators  which bind the ions essential for their action. Besides the inactivation of nucleases, proteolytic enzymes are applied during the nucleic acid isolation to remove total protein content of the sample.The most widely used proteolytic enzyme in nucleic acid purification is the Proteinase K, which was described in 1974 . ProteinCell biology studies frequently require the dissociation of primary tissues and the isolation of viable cells for tissue culturing. The most common method for cell isolation is the enzymatic digestion of the junctions connecting the cells and the components of the surrounding extracellular matrix, by which the cells can be released from a wide variety of tissues. Several enzymes are available in the market for the detachment of cultured cells, cell dissociation and cell component or membrane-associated protein isolation ,38. BesiThe experimental conditions of cell isolation are functions of several parameters, including the type of the tissue and the source of its origin. Cells with high viability can be isolated in high yield using a suitable enzyme or the optimal combination of enzymes. As proteases differ in their specificities, different enzymes are recommended to be used use for most effective tissue disruption, depending on the origin and type of the tissue. We describe below the enzymes most commonly used for cell isolation.Clostridium histolyticum, and it is capable of digesting collagen fibers very effectively. Solutions supplied for tissue dissociation contain collagenase and other additional proteinases which can digest the components of the extracellular matrix [Carica papaya latex. Papain, similarly to elastase, also has amidase and esterase activities and has a broad specificity. Papain has less damaging effects on tissues and therefore it is typically applied for cell dissociation of neuronal tissues. Besides cell dissociation, papain is also widely used for integral membrane protein solubilization and for digestion of proteoglycans. The serine protease trypsin is a very specific proteinase cleaving the peptide bonds at the C-terminal end of positively charged Lys and Arg side chains. Due to the high specificity of trypsin the digestion of tissue proteins is less effective and it is generally used for tissue dissociation together with other proteolytic enzymes. Serine protease chymotrypsin cleaves peptide bonds preferentially at the carboxyl side of aromatic Tyr, Trp and Phe residues. Chymotrypsin is less widely used for tissue dissociation; the use of other additional proteases is required for efficient digestion. The Zn-metalloprotease dispase is also a neutral protease. This non-specific protease cleaves the peptide bonds of proteins at the amino side of non-polar amino acid residues. The matrix metalloproteinase collagenase was first isolated in 1953 . This enr matrix ,40. The Cells isolated from a tissue can be cultured separately from the organism in cell culture flasks using appropriate growth medium. Adherent cells grown in a cell culture flask are attached to the surface by protein bridges which have to be disrupted during passaging. The cells can be released from the cell flask surface mechanically using a cell scraper or can be detached by a protease treatment using trypsin solution.Trypsinization means the process used for the detachment of adherent cells using trypsin solution to digest the adhesion molecules by which the cells are attached to the surface of the culture flask. Trypsin solutions generally contain EDTA to reduce the concentration of metal ions that might inhibit trypsin.Although trypsinization is the most commonly used method to detach adherent cells from cell culture flasks, the effects of this protease treatment were only recently studied in detail. It was found that trypsinization can affect the extracellular matrix surrounding the cells  and has H and VL) and constant  regions, respectively. The VH and VL chains, containing hypervariable regions, are responsible for the antigen-antibody interactions and determine the antigen specificity [Antibody molecules are produced by the immune system against foreign substances and are classified into the immunoglobulin superfamily of the proteins A. They ccificity .Fragments of the monoclonal antibodies are widely used in diagnostics, therapeutics and in biopharmaceutical research ,45,46 haH1 and CH2 domains: one Fc  and two identical Fab (antigen binding) fragments are released (2 fragment containing both antigen binding sites. The cysteine protease ficin can release both F(ab')2 or Fab fragments 2 or “IgG-like” fragments which have beneficial properties compared to the whole IgM molecules. Trypsin and pepsin are useful enzymes for IgM fragmentation, pepsin can generate Fab and F(ab')2 fragments, while trypsin can generate “IgG-like” fragments, as well. Papain cannot be used for IgM fragmentation because of the production of heterogeneous fragments. The 2 fragments [Digestion of monoclonal antibodies by papain or pepsin is still used to produce Fab or F(ab')ragments . There aragments .in vivo applications compared to whole antibody molecules. Due to their smaller size they have higher mobility and can penetrate tissues or permeate cell membranes more easily. As antigen-binding fragments lack the Fc fragments, they have lower immunogenicity, as they contain only their antigen-binding domain sites 2 fragments) and do not carry the regions responsible for antibody effector functions, furthermore, they do not form large immunocomplexes.Antibody fragments have several beneficial properties for The antigen-binding fragments of antibodies have great importance from the viewpoint of clinical and therapeutic applications. Antibody fragments could be administered to prevent the development of a disease , could be applied during the diagnosis  or for the treatment of some diseases  or to detect toxins or neutralize snake venoms ,45. The Crystallographers have great challenges in the structure determination of transmembrane proteins. It is difficult to crystallize these proteins due to their high molecular flexibility, hydrophobic surfaces and low solubility. Antibody fragments are very useful tools for solving these problems. Specific binding of antibody fragments can increase the overall hydrophilicity of proteins and the solubility of the transmembrane proteins, furthermore, they can decrease the flexibility and stabilize the conformation of the molecule . The strThe proteases have great indirect significance in structure determination, because the antibody fragments produced by proteolytic cleavage of whole immunoglobulins can be used to improve the crystallization properties ,53,54.The proteases are not only tools for crystallographers but are also important target molecules for structural biologists and have great relevance in antiviral therapies, drug and therapeutics development from the viewpoint of structural biology. Besides interest in proteases with unknown structure, the results of structural studies can help researchers evaluate the structure-function relationships more efficiently; increasing knowledge on the structural organization of viral proteases can help to explore their action, perform comparative studies by which we can better understand the structure-function and evolutionary relationships and recognize general or specific features ,56,57. OThe proteins produced by recombinant techniques are typically linked with a fusion partner termed a fusion tag. The introduction of a fusion tag means the fusion of an additional protein or peptide to the recombinant protein. These fusion tags are extensively used from basic research to high-throughput structural biology owing to the several advantages they provide in the expression of different recombinant proteins ,66. The 6), FLAG-tag, maltose binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), small ubiquitin-like modifier (SUMO), ubiquitin (Ub) and green fluorescent protein (GFP) [Commonly used affinity tags and fusion protein partners are the hexahistidine-tag (Hisin (GFP) .In some cases the tag can be removed by a chemical treatment but those methods are rather unspecific compared to enzymatic cleavages and may lead to protein denaturation and/or side chain modifications of amino acids in the target protein. The specificity and detergent sensitivity of common proteases used for tag removal have been examined and reviewed previously ,71,72,73E. coli [Serine proteases such as enterokinase , factor Xa and thrombin have been widely used for many years to remove N-terminal tags, but several cases have been reported in which they cleaved not only at the desired cleavage site but also in the protein of interest. These incidents led to the extensive application of viral proteases like human rhinovirus (HRV) 3C protease and tobacco etch virus (TEV) protease. While sequences recognized by a cellular serine protease and the viral proteases could be similar, the viral proteases cleave the protein substrates at the undesired sites less efficiently due to their high specificity and low catalytic rate, moreover, recombinant viral proteases can be produced in high quantities in E. coli . These fE. coli , West NiE. coli  and someE. coli .6 tag from the C-terminal end of a protein [Aminopeptidases and carboxypeptidases are not as widely used as endopeptidases, as they frequently leave amino acid residues on the target protein, while it is easier to design cleavage sites with endopeptidases not to leave extra residues after the cleavage. However, if is still desired to add a tag onto the C-terminal of the target protein a carboxypeptidase may be used for its removal. Among metallocarboxypeptidases, type A carboxypeptidases remove mostly aromatic or branched aliphatic side chain containing amino acids, while type B carboxypeptidases prefer basic amino acids. Carboxypeptidases can be utilized to remove a His protein . DipeptiProteomic studies are made with the aim to identify, characterize, and quantify the required samples and typically involve mass spectrometric (MS) analysis. Besides determination of the composition of protein complexes, chemical properties, post-translational modifications and structural properties of proteins can also be revealed by MS.m/z) ratio. Generally, the samples to be analyzed contain a mixture of various proteins and/or polypeptides that have to be separated and digested into smaller fragments before the MS analysis. Protein separation can be performed efficiently by polyacrylamide gel electrophoresis. The separated proteins can be digested after the electrophoresis by chemical cleavage or by enzyme-catalyzed digestion of peptide bonds. The process in which the bands or spots are cut out from the gel followed by the addition of protease(s) to the gel containing the protein(s) of interest is called in-gel digestion [MS is a powerful analytical method to measure the mass of proteins or peptides by the analysis of mass-to-charge ratio .Several proteases are available for this fragmentation of which specificities are well established ,80. The Generally, highly sequence-specific proteases are preferred for protein fragmentation instead of less specific enzymes, as the latter ones produce a very complex mixture of fragments. In the case of the efficient fragmentation the peptide, the fragments have a proper length and are released in high yield, and the complete sequence of the whole protein can be covered by the analysis of the proteolytic fragments.Besides extended application for nutritional and pharmaceutical purposes, proteases from natural sources are also widely used tools in molecular biology practice. Their degradative properties make them useful for general protein digestion in tissue dissociation, cell isolation, and cell culturing. The specificity and the predictability of cleavages by proteases enables their use for more specific tasks such as antibody fragment production, the removal of affinity tags from recombinant proteins and specific protein digestion in the proteomics field mainly for protein sequencing. Moreover, the already mentioned specificity makes proteases—in a water restricted environment—able to synthesize the peptide bonds instead of hydrolyzing them. This property combined with their enantioselectivity has also promoted their use in peptide synthesis.The expansion of knowledge has assisted the increase of applications of proteolytic enzymes for several purposes, and the application fields are widening with the help of protein engineering techniques and by chemical modification of the enzymes ,62. Stud


Hemodynamic management in intensive care patients guided by blood pressure and flow measurements often do not sufficiently reveal common hemodynamic problems. Trans-esophageal echocardiography (TEE) allows for direct measurement of cardiac volumes and function. A new miniaturized probe for TEE (mTEE) potentially provides a rapid and simplified approach to monitor cardiac function. The aim of the study was to assess the feasibility of hemodynamic monitoring using mTEE in critically ill patients after a brief operator training period.In the context of the introduction of mTEE in a large ICU, 14 ICU staff specialists with no previous TEE experience received six hours of training as mTEE operators. The feasibility of mTEE and the quality of the obtained hemodynamic information were assessed. Three standard views were acquired in hemodynamically unstable patients: 1) for assessment of left ventricular function (LV) fractional area change (FAC) was obtained from a trans-gastric mid-esophageal short axis view, 2) right ventricular (RV) size was obtained from mid-esophageal four chamber view, and 3) superior vena cava collapsibility for detection of hypovolemia was assessed from mid-esophageal ascending aortic short axis view. Off-line blinded assessment by an expert cardiologist was considered as a reference. Inter-rater agreement was assessed using Chi-square tests or correlation analysis as appropriate.P < 0.0001). There was a significant correlation between the FAC measured by ICU operators and the reference  < 0.0001).In 55 patients, 148 mTEE examinations were performed. Acquisition of loops in sufficient quality was possible in 110 examinations for trans-gastric mid-esophageal short axis, 118 examinations for mid-esophageal four chamber and 125 examinations for mid-esophageal ascending aortic short axis view. Inter-rater agreement (Kappa) between ICU mTEE operators and the reference was 0.62 for estimates of LV function, 0.65 for RV dilatation, 0.76 for hypovolemia and 0.77 for occurrence of pericardial effusion (all Echocardiographic examinations using mTEE after brief bed-side training were feasible and of sufficient quality in a majority of examined ICU patients with good inter-rater reliability between mTEE operators and an expert cardiologist. Further studies are required to assess the impact of hemodynamic monitoring by mTEE on relevant patient outcomes. Hemodynamic monitoring plays an important role in the management of intensive care unit (ICU) patients. Monitoring using a pulmonary artery catheter has been questioned due to the invasive nature of the method and the lack of clear evidence for improved outcomes associated with its insertion and use to guide therapy . SeveralEchocardiography has been established as a tool to evaluate the causes of hemodynamic instability in ICU patients by the visualization of cardiac chambers, valves and pericardium and cardiac functional abnormalities ,5. Transet al. the feasibility of hemodynamic monitoring and safety of mTEE were demonstrated in a group of 94 ventilated critically ill patients [Recently, a new technology using miniaturized probes for continual or repeated prolonged monitoring using TEE (mTEE) has been introduced. This approach potentially provides a robust, but more rapid and user-friendly approach to monitoring hemodynamic status and cardiac function than conventional TTE and TEE. In a recently published study by Vieillard-Baron patients . In thisThe objective of the study at hand was to assess the ability of novice operators with no previous experience in TEE to efficiently conduct hemodynamic assessments in an ICU setting. We systematically evaluated the image quality and inter-observer reliability during the introduction of the mTEE monitoring system in the ICU of a tertiary care center.This study is based on data collected during the prospective process quality audit in the context of the introduction of mTEE for clinical use in the Department for Intensive Care Medicine at the University Hospital of Bern, Switzerland and adheres to the tenets of the Declaration of Helsinki. The study protocol was submitted to the Ethical Committee of the Canton of Bern; the need for ethical approval was waived provided that purely observational data were collected in conjunction with the process quality audit. Nevertheless, all patients admitted to the Bern University Hospital are routinely informed of their right to specify whether data related to their stay can be used in observational studies; data of patients who declined were not included in the study.® probe is a miniaturized, 5.5 mm monoplane TEE probe . The probe produces standard monoplane  two-dimensional images; anteflexion and retroflexion of the tip of the probe is possible. The probe is approved to remain in situ for 72 h. It is connected to a dedicated ultrasound system which allows for recording of digital loops and performance of basic two-dimensional measurements of areas and distances.The ImaCor ClariTEEData were collected between February 2012 and August 2012 for the first 55 patients in whom the device was used. Patient population included both medical and surgical patients. Only patients on mechanical ventilation with an orotracheal tube in place were examined. Ventilator settings routinely used in our department consist of volume controlled immediately after intubation and pressure support thereafter. Ventilator settings were not changed for the purpose of mTEE assessments; the department's standard sedation protocol was used to avoid patient-ventilator dyssynchrony. The exclusion criteria for the use of mTEE in our institution are as follows: unrepaired tracheoesophageal fistula, history of prior esophageal surgery, esophageal obstruction or stricture, esophageal varices or diverticulum, perforated hollow viscus, gastric or esophageal bleeding, vascular ring, aortic arch anomaly with or without airway compromise, oropharyngeal pathology, severe coagulopathy, and cervical spine injury or anomaly. The decision to use mTEE for hemodynamic monitoring was made by the treating staff specialist on clinical grounds.Fourteen ICU staff specialists - none of whom had received any training in TEE before the study - received a total of six hours training in the use of mTEE by an experienced operator. Training included a 60-minute introduction of the method and demonstration of device use in the context of a presentation, followed by five hours of one-to-one bedside training in small groups of two to three trainees during the same day. Training included all necessary skills to use the mTEE device, and to acquire and interpret images according to predefined parameters. The mTEE examinations performed during the training period were not included in the analysis for the study at hand. At the time of the study, TEE examinations in our department were exclusively performed by an on-call cardiologist and not by intensive care staff; whereas 4 of the 14 operators assessed in the study had experience in TTE examination.The decision to use mTEE for hemodynamic monitoring was at the discretion of the staff specialist responsible for the treatment of a patient and was not mandated by the study protocol. After positioning of the mTEE probe, three standard TEE views  were acqAfter image acquisition the operator completed a questionnaire on the patient's hemodynamic condition based on the mTEE information. This included rating of systolic LV function , rating of RV size as dilated or not dilated, and presence or absence of hypovolemia and pericardial effusion. In addition, operators were asked to evaluate the difficulty to acquire the mTEE views  and the utility of the information obtained by mTEE and to record if any changes in hemodynamic management  were made on the basis of the acquired mTEE data. An independent observer (study nurse) recorded data during the mTEE examination regarding the successful use of the system and time from start of the system set-up at the bedside to completion of the questionnaire regarding the patient's hemodynamic condition.All recorded mTEE views and measurements were assessed off-line by an expert cardiologist blinded to the patients and the mTEE operator's identity and to the results of the operator's examination. The mTEE views were assessed for adequate quality to measure FAC , RV/LV ratio  and superior vena cava collapsibility index . The cardiologist measured LV areas at end-systole and at end-diastole and calculated FAC for every patient and answered the same predefined questions about the hemodynamic condition based on the recorded mTEE views .P-value of < 0.05 was considered statistically significant. Standard statistic software was used .Continuous variables are presented as mean (standard deviation) or median (interquartile range). Normality testing was performed using the Kolmogorov-Smirnov test. For comparison of mTEE measurements of ICU staff specialists and the cardiologist inter-rater reliability for categorical variables was assessed using a Chi-square test. Inter-rater agreement (kappa statistics) was determined. Pearson's r (1-tailed) was used to determine inter-rater reliability for continuous variables (FAC). A A total of 148 examinations were performed in 55 patients  during the study period, according for 2.7 ± 1.9 examinations per patient. Indication for mTEE assessment was circulatory failure in 45 patients  and respiratory failure in 5 patients  whereas 5 patients had other, miscellaneous indications. The mean duration of mTEE monitoring period in which the probe was left in place was 28 ± 16 hours.All ICU staff specialists were able to use the system correctly and to store images and video loops after initial training. Median time from start-up of the device until completion of examination and recording of results was 23 minutes IQR 15 to 28) if the mTEE probe was already in place and 25 minutes (IQR 20 to 33) if the mTEE probe had to be inserted. Insertion and/or acquisition of mTEE images required removal of the nasogastric tube in nine patients (16%) only for the initial placement and in six patients (11%), repeatedly accounting for the necessity of removal in 24 (16%) of all 148 examinations. Acquisition of the loops was considered easy in approximately half of the examinations and not possible in 9 to 10% of the examinations , RV dilatation  hypovolemia  and occurrence of pericardial effusion  was substantial. There was a significant correlation between the FAC measured by ICU operators and the cardiologist  < 0.0001)  examinations. A total of 56 changes in hemodynamic management after mTEE assessment of the respective patient were reported in 50 (34%) examinations. Management changes included administration of fluids in 19 instances (13%), start or increase of inotropes/vasopressors in 23 (16%), stop or decrease of inotropes/vasopressors in 10 (7%) and drainage of pericardial effusion following 4 examinations (3%).Our study shows that the use of mTEE by operators not previously trained in TEE can be introduced successfully in an ICU setting of a tertiary hospital. Echocardiographic examinations by operators using mTEE after six-hour bedside training were technically feasible and of sufficient quality in a majority of examined ICU patients. Inter-rater reliability between assessment by ICU mTEE operators and a trained cardiologist was substantial. Hemodynamic assessment using mTEE might, therefore, provide a valuable alternative to standard TTE or TEE-examination or conventional hemodynamic monitoring for a rapid, semi-quantitative assessment of LV and RV function and volume status.in situ for up to 72 hours. Our results are comparable with a recently published study by Vieillard-Baron et al. who evaluated the hemodynamic monitoring capability and safety of mTEE in a group of 94 ventilated critically ill patients [We evaluated a monoplane, oral mTEE probe for hemodynamic monitoring in a population of critically ill patients in our ICU. Our data show that after a short bed-side training our staff specialists were able to acquire and correctly interpret relevant information on key hemodynamic parameters. The acquired information was rated as useful for hemodynamic management in a majority of patients and led to therapeutic changes in a relevant proportion. No device specific complications occurred. The time needed for system start-up and to acquire the three mTEE views and process the images was substantial, but was comparable with the insertion of central lines for hemodynamic monitoring. Additionally, the duration is partially explained by the fact that in the context of the study we required the operators to assess and record all information in a protocolized fashion for every evaluation. In clinical practice, this is not necessary and real-time monitoring of hemodynamic parameters can be performed while the mTEE probe is left patients . The autet al. [Minor self-limited gastric bleeding mechanical ulceration of the superior lip was observed in two patients. However, mTEE examinations were performed by four highly trained intensivists with extensive expertise in critically care echocardiography. The authors state that their results cannot be extended to prove the ability of novice operators with no previous experience in TEE to efficiently conduct hemodynamic assessment using the tested device. Our results now also demonstrate that novice operators can obtain valid and clinically relevant hemodynamic data with mTEE although the success rate of obtaining images in sufficient quality was lower than the reported success rate of experienced TEE operators in the study of Vieillard-Baron et al. .In an ICU setting, the ideal monitoring technique allows for fast and simple but accurate continuous measurement of key hemodynamic parameters, is of low invasiveness and does not require extensive training for its use . Such a Bedside echocardiography is considered a promising tool for the management of critically ill patients , and hasOur study has several limitations. First, we did not compare the ability of a novice ICU operator with a TEE-trained cardiologist to obtain the images with the mTEE probe. It is conceivable that the high success rate could have been further improved by a trained expert. A study design allowing such comparison during hemodynamic instability could be hampered by relevant ongoing changes during the process of hemodynamic stabilization with fluids and vasopressors. We, therefore, chose to compare the interpretation of data off-line. Secondly, the study design allowed the use of any other, conventional hemodynamic monitoring modality at the discretion of the ICU specialist in charge of the patient. We cannot exclude that some of the reported decisions to change hemodynamic management also utilized additional available parameters rather than solely those acquired by mTEE - a practice we strongly advocate in the clinical routine of any hemodynamic monitoring. In our study, although hypovolemia was detected only in 5% of examinations, fluids were given nearly three times as often. We did not record the precise indication for the fluid administration, but assume that mTEE was also used to exclude dilated RV or LV when the aim was to enhance cardiac output or peripheral tissue perfusions with fluids. The impact on patient management was not the main outcome parameter of this feasibility study and we did not compare the effectiveness of mTEE monitoring with other monitoring technologies. Finally, it has to be emphasized that we report on the first mTEE examinations of each novice operator. The method of use in the context of the introduction of this new technology potentially does not reflect the future modalities of mTEE. Increasing experience might influence the decision processes for its use, such as patient selection and examination intervals, as well as technical properties such as time requirements for and quality of mTEE studies.In summary, our study shows that hemodynamic monitoring using the mTEE-system is feasible and provides additional, valid data for the management of critically ill patients after a brief training period of the operator. Further studies are required to assess the impact of hemodynamic monitoring by mTEE on relevant patient outcomes.• Hemodynamic monitoring of ICU patients using the mTEE-system by operators not formally trained in TEE is feasible with minimal training.• Hemodynamic monitoring of ICU patients using the mTEE-system provides additional, valid data for the management of critically ill patients.• Further studies are required to assess the impact of hemodynamic monitoring by mTEE on relevant patient outcomes.® miniaturized: 5.5 mm monoplane TEE probe; RV: right ventricle; TEE: trans-esophageal echocardiography; TG mid SAX: transgastric mid-esophageal short axis view; TTE: transthoracic echocardiography.FAC: fractional area change; ICU: intensive care unit; LV: left ventricle; LVEDA: left ventricular end-diastolic area; LVESA: left ventricular end-systolic area; ME 4 chamber: mid-esophageal four chamber view; ME asc aortic SAX: mid-esophageal ascending aortic short axis view; : mTEE: ImaCor ClariTEEThe authors declare that they have no competing interests.TMM designed the study, performed the statistical analysis and drafted the manuscript. LC participated in the design of the study, assisted with the assessment of mTEE image quality and helped to draft the manuscript. HB independently assessed all mTEE examinations. DB and JW participated in the design of the study and assisted with the implementation of mTEE in our unit. JT conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors revised the manuscript for important intellectual content; and have given final approval of the version to be published.


Cleft lip and palate are craniofacial anomalies highly prevalent in the overallpopulation. In oral clefts involving the alveolar ridge, variations of number,shape, size and position are observed in maxillary lateral incisors. The objectiveof this manuscript is to elucidate the embryonic origin of maxillary lateralincisors in order to understand the etiology of these variations.  The hypothesis that orofacial clefts would split maxillary lateral incisor budshas been previously reported. However, recent studies showed that maxillarylateral incisors have dual embryonic origin, being partially formed by both themedial nasal process and the maxillary process. In other words, the mesial half ofthe lateral incisor seems to come from the medial nasal process while the distalhalf of the lateral incisor originates from the maxillary process. In cleftpatients, these processes do not fuse, which results in different numerical andpositional patterns for lateral incisors relating to the alveolar cleft. Inaddition to these considerations, this study proposes a nomenclature for maxillarylateral incisors in patients with cleft lip and palate, based on embryology andlateral incisors position in relation to the alveolar cleft. Embryological knowledge on the dual origin of maxillary lateral incisors and theuse of a proper nomenclature for their numerical and positional variations rendersappropriate communication among professionals and treatment planning easier, inaddition to standardizing research analysis. Cleft lip and palate are craniofacial abnormalities characterized by the absence ofcontinuity of maxillary structures-Cleft lip and cleft lip and palate determine a break in the alveolar ridge with animpact on the dentition and treatment prognosis.This article aims to discuss, based on an embryological analysis, variations in numberand position of maxillary lateral incisors in individuals with cleft lip and cleftpalate, in addition to presenting a proposal of nomenclature for clinical and researchusage and the clinical implications of cleft lip and palate patients'rehabilitation.,,The human face is developed during the first weeks of intrauterine life. The maxillais embryologically constructed through the fusion of medial nasal processes which arecentrally positioned on the face, in addition to bilateral maxillary processeslaterally positioned.In noncleft patients, anatomically, the maxilla consists not only of the pre-maxilla,region where the dental germs of the four incisors are located, but also of themaxillary posterior segments, where the dental germs of canines, pre-molars andmolars are located. The pre-maxilla and the maxillary posterior segments areseparated by a V-shaped incisive suture.However, in patients with cleft lip and palate, a lateral incisor is commonly foundin the posterior maxillary segment, distal to the alveolar cleft region and mesial tothe maxillary canine, i.e., out of the pre-maxilla. How this occurrence is explainedif lateral incisors are located in the pre-maxilla, mesial to the incisive suture, inpatients without clefts? To make matters worse, some patients with orofacial cleftspresent a maxillary lateral incisor mesial to the alveolar cleft with or without thepresence of a lateral incisor distal to the cleft. The question that arises is: Wouldthe alveolar cleft split the maxillary lateral incisor tooth germ into two parts?What is the explanation for the location of a lateral incisor out of thepre-maxilla?,,,In reality, there is no correspondence between the alveolar cleft location and theincisive suture .6,11,,The hypothesis of dual embryonic origin of maxillary lateral incisors wascorroborated by the study of Hovorakova et al.Corroborating the theory of dual embryonic origin of maxillary lateral incisors, astudy analyzing monkeys' embryos with oral clefts found a union between the dentallaminae of the medial nasal and maxillary processes forming an epithelial bridge,responsible for the formation of the embryonic lateral incisor tooth bud.,Considering this new evidence, the former hypothesis assuming that oral cleft coulddivide the tooth germ of maxillary lateral incisorsWith this in mind, the theory most accepted currently is the maxillary lateralincisor dual embryonic origin, with partial origin at the medial nasal process andpartial origin at the maxillary process. This knowledge makes it possible tocomprehend the high frequency of number anomalies shared by patients with clefts. Infact, there are four clinical numerical and positional situations regarding maxillarylateral incisors in the alveolar cleft region: Presence of two maxillary lateral incisors, one mesial and the other distalto the alveolar cleft .Presence of a single lateral incisor positioned mesial to the alveolar cleft. Presence of a single lateral incisor located distal to the alveolar cleft. Agenesis of maxillary lateral incisor adjacent to the cleft side : absenceThe frequency of each one of these four occurrences varies according to the type ofcleft and is compiled in ,The current nomenclature for maxillary lateral incisors adjacent to the alveolarcleft is very confusing. There are several reports in the literature on thedifficulties for research due to varied nomenclature and lack of astandardization.Some studies use the term "supernumerary" when there are two lateral incisors in theregion of the cleft. Another denomination, dating back to 1973, is the term"pre-canine" to refer to lateral incisors distal to the alveolar cleft.With the recent evidence of the dual origin of maxillary lateral incisors, the needof a new nomenclature was raised. Considering the four clinical variations of lateralincisors in patients with cleft lip and palate, as described in the previous topic,the use of the following nomenclature is proposed :» Mesial lateral incisor (12M or 22M) and distal lateral incisor (12D or 22D) - theseterms would be assigned to lateral incisors mesial and distal to the cleft,respectively. Therefore, the terms mesial and distal refer to the lateral incisorposition in relation to the alveolar cleft. In other words, this new nomenclature considers as maxillary lateral incisors anytooth located between maxillary central incisor and canine. The goal of standardizingthe nomenclature for maxillary lateral incisors is to simplify the communicationbetween professionals involved in rehabilitation of orofacial clefts as well as tomake description in clinical or laboratory research in the field of moleculargenetics easier.Agenesis is the most prevalent dental anomaly shared by cleft patients.,,The most severe cases of oral clefts present higher prevalence of tooth agenesis.,,Different clinical patterns for maxillary lateral incisors in patients with oral cleftscan be explained by the dual embryonic origin of these teeth. The presence of a singlemaxillary lateral incisor located mesial or distal to the cleft region can result indifferent degrees of hypoplasia of the embryonic processes involved in cleftformation.In ,What are the clinical implications of this new evidence and of the new proposednomenclature? Orthodontic treatment is an important step in the rehabilitation processof the individual with orofacial cleft. In patients with agenesis of the lateral incisoron the cleft side , orthodo,Patients with at least one lateral incisor, mesial (12M or 22M) or distal (12D or 22D),as illustrated in For patients with both maxillary lateral incisors (12M and 12D or 22M and 22D) on thecleft side, as shown in The location of the maxillary lateral incisor in relation to the alveolar cleft directlyinfluences the position of adjacent teeth. When the lateral incisor is mesial to thecleft, there is less space available at the pre-maxilla and, as a result, centralincisors have a greater probability of erupting with rotationsIn summary, the numerical variation of cases of maxillary lateral incisors in patientswith oral cleft have several treatment plans that present good esthetic and functionalresults. When only one half of a lateral incisor is present adjacent to cleft, itspreservation is important to maintain the alveolar bone in the cleft region, therebyimproving the bone-graft prognosis and providing a good esthetic result.The dual origin of maxillary lateral incisors stimulates new hypotheses that should beevaluated in the near future. Within this perspective, the presence of two maxillarylateral incisors in patients without clefts could represent a mild expression of cleftlip and palate. In patients with isolated cleft lip, the frequent presence of twomaxillary lateral incisors would represent an incomplete manifestation of the alveolarcleft.The dual origin of maxillary lateral incisors may justify the vulnerability fornumerical and positional clinical variations. This study proposes a nomenclature thatspecifies the position of the lateral incisor in relation to the alveolar cleft. Twolateral incisor adjacent to the alveolar cleft can be observed, one mesial lateralincisor (12M or 22M) and one distal lateral incisor (12D or 22D). The proposednomenclature contributes to the effectiveness of communication between the team duringthe rehabilitation of cleft lip and cleft palate besides standardizing researchanalysis.


Dentin dysplasia, a rare hereditary disorder of dentin formation, is characterized by normal enamel but atypical dentin formation along with abnormal pulpal morphology. It is inherited as an autosomal dominant trait. It has been divided into two clinical entities: type I (radicular) and type II . Early diagnosis and initiation of effective regular dental treatments may help the patients with this condition to delay or prevent the loss of the entire dentition and help them in cope up with edentulous state in early ages. The condition undoubtedly has a negative impact on the physical and psychological well-being of the affected individual. Numerous factors have to be considered during the prosthetic rehabilitation of patients with dentin dysplasia. Treatment protocol varies according to clinical case. Although literature reports suggest general guidelines for treatment planning, the present case report describes a full mouth rehabilitation of an 8-year-old female patient with dentin dysplasia.How to cite this article: Khandelwal S, Gupta D, Likhyani L. A Case of Dentin Dysplasia with Full Mouth Rehabilitation: A 3-year Longitudinal Study. Int J Clin Pediatr Dent 2014;7(2): 119-124. The disturbance of dentin development was recognized, such as highly atypical dentin, accompanied with obliteration of the pulp and defective root formations. Stafine and Gibilisco later reported three cases with similar radiographic findings but there were no microscopic descriptions.2Dentin dysplasia (DD) is an autosomal dominant hereditary disturbance in dentin formation, which may present with either mobile teeth or pain associated with spontaneous dental abscesses or cysts. This rare anomaly of unknown etiology, affects approximately one patient in every 100,000.Carrol O et al proposed a subclassification based on the radiographic findings. They proposed two basic types. Dentin dysplasia type I is characterized by the presence of primary and permanent teeth with normal appearance of the crown but no or only rudimentary root development, incomplete or total obliteration of the pulp chamber and periapical radiolucent areas or cysts. DD-I was classified into four subtypes: Ia, Ib, Ic and Id. Type Ia is characterized by a complete obliteration of the pulp and usually little or no root development. Subtype Ib has a horizontal, crescent shaped, radiolucent line, separating normal coronal dentin from abnormal radicular dentin and short, conical and rudimentary roots. Type Ic affected teeth shows two crescent-shaped horizontal radiolucent lines with their concavities toward each other at the cement-enamel junction. The roots are one half the normal lengths. Normal root formation, which sometimes may be bulbous in the coronal third, is seen in type Id. A stone may be found within the pulpal canal. In this least severe type of DD, normal root formation occurs and the pulp chamber is usually not obliterated. In these cases, the pulp around the stones is healthy. In other cases, the denticle is continuous with dentinal walls.3Unlike the first variant, DD type II is characterized by primary teeth with complete pulpal obliteration and brown or amber bluish coloration similar to that seen in hereditary opalescent dentin. The permanent teeth have a normal appearance or a slight amber coloration; the roots are normal in size and shape with a thistle-tube shaped pulp chamber due to sudden constriction of the chamber with pulp stones.4 This report highlights the multidisciplinary treatment approach of a female child suffering from DD-I facing almost complete edentulous state.The sequelae of dentin dysplasia are difficult to manage and provide a challenge for the dentist concerning restorative and endodontic treatment but also prosthetic treatment after loss of teeth.An 8-year-old girl reported with complaints of pain in gums and difficulty in eating since 1 year. Her most of the teeth fell down within a short period of eruption, as reported by her mother. There were no previous cases of this disturbance in the familial history and was nothing significant in her medical and personal history.General physical and intellectual examination revealed the normal findings except her discolored hair, which was similar to her mother . The intOrthopantomogram (OPG) was done to evaluate the status of erupted and developing dentition. Orthopantomogram  confirmeBased on the clinical examination and investigations, final diagnosis of dentin dysplasia type I was made.Patient was treated with using multidisciplinary approach. Based on pedodontist’s and endodontist’s view, extraction of 64 and restoration of all the decayed teeth  were planned and performed . FollowiGround section of extracted tooth under stereomicroscope revealed thickened dentin and narrow pulpal space . As rootIt is anticipated that unerupted permanent teeth may undergo premature removal as erupt into the oral cavity. The possibility of endosseous implants is being explored for when the patient reaches her late teens and growth is complete.356Little is known about etiology of DD in healthy individuals. Several factors possible causes have been implicated, but the exact mechanism of the defect is yet to be determined. Dentin contains collagen type I and II, the abnormality of which is presented as DD of different forms and severity. DD-I is an inherited disorder characterized by atypical development of the dentin. This disorder is also known as radicular dentin dysplasia as the underdeveloped, abnormal pulp tissue is predominately in the roots of the teeth. The teeth either lack pulp chambers or have crescent shaped pulp chambers with short or abnormally shaped roots. This abnormal root morphology is thought to be secondary to the abnormal differentiation and/or function of the odontoblasts. The condition may affect both the dentition and, since the roots are abnormally short, premature loss of teeth is usually seen while, the color of the teeth is usually remains normal. DD-II is characterized by primary teeth with complete obliteration of the pulp. The teeth shows brown or amber bluish coloration similar to hereditary opalescent dentin. The permanent teeth, however, have a normal appearance or a slight amber coloration, the roots are normal with a thistle-tube shaped pulp chamber with pulp stones.7Different theories have been postulated to explain the pathogenesis of the disease. The first theory states that dental papilla is responsible to the extent with multiple foci of degeneration of the same that calcify and, during the healing period, obliterate the pulp space. The second states that the early invagination of the epithelium in the embryonic process subsequently generates multiple foci of dentin formation. The third states the alteration in the normal interaction that exists between the layer of the odontoblasts and ameloblasts to be responsible for the abnormal dentin formation.78Mutations in two genes, MSX1 and DSPP, have also been thought of in the disease pathogenesis in a recent study. The DSPP gene was considered within the study as candidate gene for the etiology of the dentin dysplasia type I, due to the description in the literature of mutations in exon 2 of the DSPP gene in patients with dentinogenesis imperfecta. The mapping on chromosome 4q21.3 of the dentin dysplasia type II region in which the DSPP gene encodes also aided in recognizing this gene as the candidate gene.9 Variety of congenital syndromes, including familial calcinosis and the brachioskeletogenital syndrome and Ehlers Danlos syndrome, is a systemic condition of genetic disorders of collagen in which patients reveal skin abnormality and joint hypermobility have been reported in association with DD.56In general, the diagnostic criteria rests solely on clinical grounds; however, as the genetic mutations underlying these conditions are now being recognized, molecular genetic diagnosis may serve as an useful adjunct to clinical evaluation, particularly when there is a doubtful diagnosis. Vitamin D-dependent rickets and vitamin D-resistant rickets have clinical and radiographic features of dentinogenesis imperfecta-I and DD. Chemotherapy or irradiation of the jaws during the period of root development leads to arrested development and can give a radiographic appearance similar to DD-I.7 Maxillomandibular atrophy is a consequence of no or partial root development and early tooth loss. Successful oral rehabilitation with complete denture after extraction of all teeth and curettage of cysts has been described.10Life expectancy of dentition is minimal, although various treatment strategies, including conventional endodontic therapy, periapical curettage or preventive regimen, have been proposed to maintain the teeth as long as possible. Early exfoliation of the teeth and associated maxillomandibular atrophy is seen in DD-I.910 Until growth is complete, the treatment of choice for the replacement of missing teeth is dentures. Ridge augmentation followed by dental implants may be considered at about 18 years of age, when growth is completed.The treatment plan aims to remove the infection or pain, restores esthetics and protects posterior teeth from wear. Treatment may vary given the age of the patient, severity of the problem and the presenting chief complaint. As in the primary dentition, one of the options is overdenture in permanent too. Those with DD-I have mobile teeth due to very short roots and, as a result, there is early exfoliation of teeth in the primary and permanent dentition.9Exposed dentin is more susceptible to caries than enamel. Regular dental checkups and caries prevention by means of oral hygiene instructions, diet counseling and appropriate fluoride application is essential. Early diagnosis and regular dental check-ups however do not aid in preventing premature exfoliation.The outcome of a diagnosis of DD largely depends upon the age at which the condition is being diagnosed and the rapid treatment protocol. Where diagnosis occurs early in the life of the patient and treatment follows the above recommendations, good esthetics and function can be obtained thereby minimizing nutritional deficits and psychosocial distress.


In this study, the lateral geniculate bodies (LGB) of rats, bats and pangolins were compared using histological and quantitative histochemical parameters to observe possible modifications that enable these mammals to cope with their habitation particularly with respect to their diet. The study was conducted using ten adult Wistar rats, ten fruit bats and eight pangolins comprising of both sexes. After being sacrificed by cervical dislocation, their skulls were opened using bone forceps to expose the brains. The lateral geniculate bodies were excised from each brain tissue, homogenized and homogenate studied spectrophotometrically for the activities of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PDH), acid phosphatase (ACP), alkaline phosphatase (ALP) and acetylcholinesterase (AChE). The LGB tissue samples meant for histological studies were fixed in 10% formol calcium and processed for paraffin wax embedding. Serial sections of 3μm thickness were stained with Hematoxylin and Eosin (H & E) and Cresyl fast violet (CFV) stains. The stained tissues were studied under the light microscope. Application of one-way ANOVA statistical method showed that there were significant differences (p<0.05) in the activities of LDH, G-6-PDH, ACP, ALP and AChE of the LGB of the three mammals as revealed in the quantitative histochemistry of these enzymes and markers. Histological observations revealed no observable differences in the relative distribution of neurons and their supporting glial cells within the LGB of the three mammalian species. The comparison of the differences observed in the histological and the quantitative histochemical activities in these mammalian species revealed a variation in the visual perception and their individual peculiarities in relation to their mode and pattern of living. Visual perception plays a role in the animal kingdom, most notably for the identification of food sources, suitable habitats, predators, and mate recognition in mating processes, as well as visual functioning to orient animals in their overall ecological surroundings . In mammcorpus geniculatum, is the thalamic relay nucleus for the visual system that lies rostral and lateral to the medial geniculate nucleus, lateral to the crus cerebi, and ventral to the pulvinar  into its reduced form, NADPH, and glucose-6-phosphate is then converted into a pentose sugar (ribulose-5-phosphate). The 5-carbon sugar is a precursor of DNA, RNA, and ATP (G-6-PDH is the first and rate limiting enzyme in the pentose phosphate pathway. G-6-PDH converts nicotinamide adenine dinucleotide phosphate (NADP and ATP . LDH cat and ATP .Acid phosphatase (ACP) is a family of enzymes that belong to the hydrolase class. It possesses the ability to catalyse the hydrolysis of orthophosphate monoesters under acidic conditions  since itAlkaline phosphatase (ALP) is a group of catalytic proteins found in all body tissues sharing the capacity to hydrolyze phosphate esters in alkaline medium (Acetylcholine (ACh) is a neurotransmitter common to many synapses throughout mammalian nervous system ranging from the diffused system in the brain to the neuromuscular junctions. Acetylcholinesterase (AChE) is one of the most crucial enzymes for nerve response and function . AcetylcThis research work was therefore designed to compare differences in the histological and quantitative histochemical activities in the LGB of the mammalian species that enable them adapt and cope with their habitat despite their varying feeding habits, mode of lives and habitat differences.Rattus norvagicus), ten fruit bats (Eidolon helvum), and eight pangolins (Manis tricuspis) of both sexes were used for this comparative study. The adult Wistar rats were obtained from the animal holdings of the department of Anatomy of the University of Ilorin and sacrificed shortly after procurement. The bats were curled down from their roosting colony with the assistance of experts who possess state permit at the bats colony at the Flower Garden area of Government Reserve Area (GRA), Ilorin and sacrificed shortly thereafter. The pangolins were procured from Asejire, a village in the North West Area of Osun State and sacrificed before dark the same day. All experimental procedures followed the recommendations provided in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institute of Health . The G6-PDH, LDH and ACP kits were purchased from Randox laboratory limited, UK, ALP from Quimica Clinica Aplicada’s, Spain and AChE kit from AGAPPE DIAGNOSTIC LTD, UK.Ten adult Wistar rats  and assayed within 48 hours. Through spectrophotometry, the activities of G-6-PDH, LDH, ACP, ALP and AChE were determined in the homogenate by the methods of Values were reported as mean ± SEM (standard error of the mean). Significance was determined statistically by application of one-way analysis of variance (ANOVA) using statistical software SPSS version 17 at 95% confidence interval. Differences between means were considered statistically significant at P<0.05.It was observed in the sections stained in H & E and demonstrated in the photomicrographs labeled Using Cresyl Fast Violet (CFV) staining technique to compare the abundance of Nissl substance of the mammalian species as demonstrated in the photomicrographs labelled Result obtained from the present study showed significant differences in the activities of the enzymes studied using quantitative histochemistry .Comparatively, the histological observations of the lateral geniculate body (LGB) of the three mammalian species stained with H & E method illustrated in Nissl bodies are strongly basophilic inclusions found in the cell bodies of neurons. These granules are rough endoplasmic reticulum with free ribosomes and are the sites of protein synthesis. Increased Nissl substances as evidenced in Cortical Structures including lateral geniculate body have higher glucose utilization than other anatomical structures . GlucoseThe measure of the quantitative histochemical activity of G-6-PDH in LGB in the three mammalian species showed significant variations. LGB of rat employed the largest amount of G-6-PDH activity; hence is the most likely using the largest quantity of energy to mediate vision and visual perception. This is followed by pangolin while the bat had the least.In overexertion conditions when oxygen is absent or in short supply to cope with energy demands of the LGB, LDH comes in to play in energy production. From the result obtained, LDH in rat LGB had the highest the quantitative histochemical activity, followed by the bat and the least was observed in the pangolin. The rat LGB utilizes the highest base on its energy demand to cope with lifestyle. Bat engages in true flight and employs an alternative pathway for its metabolism. This suggests why bat have a higher activity level than pangolin. A closer look at the G-6-PDH:LDH in examining carbohydrate metabolism as a balance between aerobic and anaerobic metabolism shows;The ratio of the activities of G-6-PDH:LDH in rats indicate that the anaerobic system is more active than the aerobic system, while the same was observed in the Bats with the levels of LDH being higher than those of G-6-PDH indicating a negative shift in oxygen usage for metabolism, while a positive shift was seen in the pangolin.The measure of ACP activity was found to be higher in bat LGB, followed by rat and pangolin has the least. The higher ACP activity in bat LGB may indicate higher amount of unused substances in form of precursors of neurotransmitters within the lateral geniculate nuclei mediating vision and visual perception. It also help to “mop up” the phosphate ions produced as a result of ATP hydrolysis.The measure of ALP activity was however found to be higher in the LGB of pangolin followed by bat and the least being rat. Pangolins are more likely generating more free energy through ALP to compliment for their LGB metabolic process. ALP mainly facilitates transport across cell membranes, causing the breakdown of ATP to ADP and inorganic phosphate, thereby making free energy available for metabolic process (The measure of the cholinergic neurons was compared in the LGB of the mammalian species. It was found that the rat LGB uses higher amount of AChE; hence employs most of this neurotransmitter to mediate neural impulses, followed by bat and then pangolin. AChE is the neurotransmitter common to many synapses throughout mammalian nervous systems. Depending on the type and function of a particular neuron, neurotransmitters may cause or inhibit the transmission of neural impulses. The physical and neurochemical characteristics of each synapse determine the strength and polarity of the new input signal.The measure of the histological observations and quantitative histochemistry in the lateral geniculate body of the mammalian species confirms that the variations observed were due to their individual peculiarities. The feeding habits, lifestyle as well as the nature of their habitat would account for this. While rats are active during the day and night with higher mobility, pangolins are only active at night. However, being an insectivore, they require highly effective vision to identify and contrast food which could be quite small, although, olfactory communication also plays a significant role (The histological and quantitative histochemical results revealed a relationship between the visual perception and specie peculiarities of the mammalian species in relation to their mode and pattern of living. This however maybe suggested as the reason for the difference in the feeding and habitat pattern of the studied mammalian species.


Photoplethysmography (PPG) is a technique that permits noninvasive measurement of changes in the volume of tissues. A novel device uses PPG to assess changes in duodenal mucosal perfusion. When tested in septic piglets, data obtained using this device correlate with the blood lactate concentration and duodenal serosal microvascular blood flow as measured with a laser Doppler flowmeter. This new PPG-based approach for continuously monitoring gut mucosal perfusion warrants further development, leading to prospective clinical trials in patients. Critical Care, Jacquet-Lagrèze and colleagues from several institutions in Lyon, France, report results from a preclinical study of a novel perfusion monitoring device [Pseudomonas aeruginosa to induce septic shock; the remaining (control) animals were not challenged with the Gram-negative bacteria preparation.In the previous issue of g device . The stuThe novel monitoring device was developed by Advanced Perfusion Diagnostics , a biotePPG and its applications in medicine have been well described in an excellent review article by Allen . PPG useThe characteristic signal from a PPG device is a waveform. The dominant  aspect of the tracing is synchronized with the beating of the heart, and is usually called the alternating current (AC) component. The origins of the other components of the PPG signal remain to be completely elucidated, although important factors are recognized to be changes in circulating blood volume, respiration and vasomotor tone. The quasi-static component of the PPG signal is usually called the direct current (DC) component.The device, which was evaluated by Jacquet-Lagrèze and colleagues, features a reflectance type of PPG device fitted onto the surface of a balloon located near the distal end of a small bore feeding tube. When the end of the feeding tube is advanced through the pylorus into the duodenum and the balloon is inflated, the PPG element presses against the mucosal surface of this portion of the intestine, allowing continuous monitoring of the AC and DC components of the waveform. In their studies using pigs, the research group from Lyon showed that the variations from baseline in both the AC and DC components of the duodenal mucosal PPG signal were significantly correlated with the variations from baseline in duodenal serosal microvascular blood flow measured using laser Doppler flowmetry. Importantly, the AC and DC components of the PPG signal also were significantly correlated with the blood lactate concentration. The findings from this preclinical validation study thus support the view that the PPG-based duodenal mucosal blood flow monitoring device provides meaningful data.While the report by Jacquet-Lagrèze and colleagues should provide encouragement for the company that is developing the new monitoring device, a look back at the history of this field should temper their enthusiasm and ours. As pointed out by the authors, use of another gastrointestinal perfusion monitoring device – the gastric tonometer – was shown to significantly improve survival in a subset of critically ill patients . But theDuodenal mucosal perfusion monitoring, using a cleverly designed PPG-based device, warrants further development, ultimately leading to prospective clinical trials. When these trials are designed, it will be important to pay careful attention to the intervention(s) that are triggered when the device indicates that intestinal mucosal perfusion is low.


Diabetic foot complications are the most serious and costly problem of Diabetes Mellitus affecting substantially the quality of life of these patients. People with diabetic foot ulcers experience severe restrictions on daily life as a result of the ulcer. They face social isolation due to reduced mobility, they require frequent clinical treatment and constant caution to ensure that effective care is taken of the feet.The primary aim of this study was to compare the patient perceptions of their quality of life in two moments of the timeline of the diabetic vascular disease: pre revascularization surgery period and post amputation.An exploratory cross-sectional study was done in a sample of 28 patients with peripheral vascular complications of diabetes, divided in two groups of 14 individuals. One group consisted of outclinic patients with lower limb amputation  for at least six months. Another group consisted of hospitalized patients with critical ischemia of the lower limbs, waiting for revascularization procedures. The Portuguese version of the Medical Outcomes Study 36 -Item Short Form (SF- 36) and the Brazilian version of the Problems Areas in Diabetes Scale (B-PAID) questionnaires were used.Patients who had been through amputation had significant (p< 0.05) lower impairment in the total score , vitality , role emotional  and mental health  domains than pre revascularized patients. The statistical difference between groups regarding the B-PAID questionnaire score was significant (p<0.01), with the best Results showed by patients with amputation .Those findings demonstrate that diabetic patients with critical ischemia in the lower limbs had worst quality of life scores than those with amputation. The postponing of an amputation must be well judged when a critical ischemia of the lower limbs is seriously impairing the patient's quality of life.