Neurotrauma or injuries to the central nervous system (CNS) are a serious public health problem worldwide. Approximately 75% of all traumatic brain injuries (TBIs) are concussions or other mild TBI (mTBI) forms. Evaluation of concussion injury today is limited to an assessment of behavioral symptoms, often with delay and subject to motivation. Hence, there is an urgent need for an accurate chemical measure in biofluids to serve as a diagnostic tool for invisible brain wounds, to monitor severe patient trajectories, and to predict survival chances. Although a number of neurotrauma marker candidates have been reported, the broad spectrum of TBI limits the significance of small cohort studies. Specificity and sensitivity issues compound the development of a conclusive diagnostic assay, especially for concussion patients. Thus, the neurotrauma field currently has no diagnostic biofluid test in clinical use.We discuss the challenges of discovering new and validating identified neurotrauma marker candidates using proteomics-based strategies, including targeting, selection strategies and the application of mass spectrometry (MS) technologies and their potential impact to the neurotrauma field.Many studies use TBI marker candidates based on literature reports, yet progress in genomics and proteomics have started to provide neurotrauma protein profiles. Choosing meaningful marker candidates from such ‘long lists’ is still pending, as only few can be taken through the process of preclinical verification and large scale translational validation. Quantitative mass spectrometry targeting specific molecules rather than random sampling of the whole proteome, e.g., multiple reaction monitoring (MRM), offers an efficient and effective means to multiplex the measurement of several candidates in patient samples, thereby omitting the need for antibodies prior to clinical assay design. Sample preparation challenges specific to TBI are addressed. A tailored selection strategy combined with a multiplex screening approach is helping to arrive at diagnostically suitable candidates for clinical assay development. A surrogate marker test will be instrumental for critical decisions of TBI patient care and protection of concussion victims from repeated exposures that could result in lasting neurological deficits. A general goal of "proteomics" is to comprehend the relationship between the body’s proteins and how they change by disease to understand human pathophysiology, and ultimately to provide therapeutic and diagnostic tools. The completion of the human genome provided researchers with the blueprint for life; proteomics offers the potential means for analyzing the expressed genome. Proteomics attempts to determine how genes function within the genome and how they communicate with each other to (hopefully) lead to important new insights into disease mechanisms. The potential of proteomics to advance biomedical research is high because the key functional components of biochemical systems and the cellular targets of therapeutic agents, namely proteins, are being studied. Mapping proteomes from injured tissues, cells and biofluids can potentially reveal new protein targets to explore mechanisms of insults and to provide candidate lists for new disease indicators or injury biomarkers as diagnostic or prognostic tools for the clinic.A biomarker could be simply a molecule, such as a protein whose presence or abundance in a biological sample signals a disease or insult to an organ. Thus, they are quantifiable molecules that indicate a pathophysiological process. A biomarker in accessible body fluids or tissues could greatly enhance our ability to identify patients at risk, with invisible wounds or predict outcome of serious injury. A sensitive and specific disease or injury marker such as an early protein abnormality could provide a warning sign prior to being symptomatic, and hence could result in more effective preventative care or treatment options to improve outcome.The goal of clinical proteomics to discover new disease or injury biomarkers is challenging. Beyond the number of human genes coding for proteins, proteins are processed and modified, comprising an important dimension of information to which present proteomic technologies have but limited access. The total mRNA population, accounting for alternate splicing, RNA editing, and use of alternate promoters could contain 250,000 transcripts, while various protein modifications could increase the size of the human proteome to over 500,000 members. CellulaDespite all of the significant advances in technologies in proteomics since its inception in the mid-1990s, with the development of more sensitive mass spectrometry detectors and more selective and specific strategies for sample processing and handling, no clinically validated disease biomarker has been discovered by proteomics to date.analytical challenge of the large number of plasma proteins lays in their unbalanced abundance: albumin constitutes over 50% of the plasma proteins (at 30–50 mg/mL) and the most abundant 22 proteins in plasma represent approximately 99% of the total protein content in plasma leaving the majority of proteins at very low abundance. The estimated dynamic range of protein concentrations in human plasma may be up to 12 orders of magnitude[What are the major factors that hindered finding robust disease and injury biomarkers and how can these be overcome? The complexity of clinical samples themselves is a significant limiting factor. Plasma and serum, i.e., blood, have been biofluids of choice for measuring levels of proteins and other biomolecules for clinical testing, as they can be sampled noninvasively. Plasma is a protein-rich information source containing what blood circulation has encountered on its journey throughout the body and tissue perfusion. The tremendous Disease or insults trigger acute events, secondary and chronic sequelae, including inflammation, wound healing, and adaptive changes that the compromised body undergoes in response to the unhealthy state. In an effort to identify original disease causes or injury factors a simple experimental model can facilitate a targeted screen circumventing secondary, less disease-specific events. As such, scientific experimental model design follows controlled strategies for reproducibility and simplicity that can facilitate the initial discovery by limiting candidate markers to those proteins that are related to a disease origin or injury cause,5. One cA second strategy advocates first enzymatically  or chemically cleaving ("breaking") a complex mixture of cellular proteins, and then "sorting" the peptides by one or more steps of chromatography. MS analyzes the recovered fragments as in the previous approach, and software matches the fragments to the proteins from which they are derived. Examples of this experimental approach include multidimensional protein identification technology (MudPIT) that couples two or more dimensions of chromatographic separations, e.g., strong cation exchange (SCX) with reversed-phase chromatography,10. Whilselection of suitable marker candidates from the ‘long list’ of identified injury or disease specifically changed proteins should arrive at a manageable ‘short list’ of possible disease marker candidates. A tailored selection strategy will consider injury cause, marker candidates with the necessary reporting power for the cause as well as organ specificity and exclusion of proteins normally present in healthy plasma and tissues.A straightforward The subsequent validation of selected disease or injury markers from a group of candidates may occur stepwise starting with a preclinical smaller cohort of patients and controls, allowing to test for normality. Followisignature injury of the wars in Iraq and Afghanistan compared to past wars[mild TBI (mTBI), many without visible wounds that often are undiagnosed[Better diagnostic tools are needed to detect head injuries, especially mTBI, to confirm and to monitor the severity of TBI in order to determine the best course of action acutely and later post-injury. The neurotrauma field has currently still no chemical diagnostic marker in clinical use. Here we will outline briefly the spectrum of TBI and give examples where a surrogate chemical marker assay for TBI would be of great benefit to patients, high risk populations, their families and doctors.Neurotrauma to the central nervous system (CNS) is a serious public health problem in the US; among US civilians, TBI is most common in infants and toddlers, adolescents and the elderly. The US ast wars. TBI condiagnosed. Better Head injuries can be classified into penetrating and non-penetrating TBI. Penetrating TBI involves physical compromise of the skull by an external object resulting in specific, focused injury most commonly characterized by hemorrhages and lesions. Non-penetrating TBI, is much more difficult to assess, as injuries may not be visible or located precisely. Closed head injuries are caused by rapid acceleration and deceleration of the brain within the skull and inflict shear and deformation forces on gray matter tissue and white matter tracts. Each trDiagnostic neurotrauma tools include imaging techniques, neurocognitive examinations, and for severe TBI patients, the determination of post-traumatic amnesia, but they provide only estimates of the dynamically evolving injury process. Functional MRI (fMRI) and the detection of regional blood flow changes  are not always available, cannot be obtained in critically ill patients, and are not definitive. Radiological brain scans on infants and toddlers are widely considered problematic because the radiation dose endangers the developing brain. Absence of imaging in the pediatric clinical praxis prevents distinguishing brain injury from frequent intestinal flu or even infant irritability. Non-accFor the vast majority of mTBI/concussion patients, there are no objective diagnostic or prognostic tools. A readysecond hit to a concussed, vulnerable brain can, in rare cases, have a catastrophic outcome with permanent brain damage or even death (known as the second impact syndrome)[Moreover, a yndrome). Severalyndrome),30. Miliyndrome),32.Certain areas of the brain may be more susceptible to concussive trauma. A recent study investigated longitudinal changes in global and regional brain volume in patients one year after mTBI and correlated such changes with clinical and neurocognitive metrics. Magnetic resonance imaging data showed measureable global brain atrophy, larger than that in control subjects one year after mTBI. Atrophy was found in specific regions of the cingulate cortex independent of the site of initial trauma. The cingulate cortex’s role in rational cognitive functions such as empathy, impulse control, and emotion correlate strongly with the patient’s observed clinical symptoms of increased depression and anxiety. These fCurrent evaluation of concussion is basically an assessment of neurocognitive deficits, often not immediate and requires extensive neuropsychological testing that is subject to motivational confounds, while critical care treatment decisions have to be made immediately by emergency clinical personnel and surgeons. Severity classification of TBI patients relies on assessing the level of consciousness, commonly with the Glasgow Coma Scale (GCS), which is an insensitive measure. Testing relies on verbal communication, and proper motor control and eye function, which are often impaired after TBI. Brain function-altering substances such as drugs, alcohol, pain medication, sedatives, or even induced coma as part of emergency and intensive care routine obviously compromise the use of memory recall and the GCS. Although predictors of TBI exist, such as the Standardized Assessment of Concussion test, these tools offer little insight into the pathology of the disease beyond determining whether a concussion has occurred or not. Because of this lack of insight into TBI, licensed health care providers of concussive sports injury are conservative in their approach to player safety after injury with the hope that coordination between sideline and clinical practitioners will aid in improving our understanding of the extent of impairment for various types of sports related concussions.An ideal biomarker should be both specific to head trauma as well as sufficiently sensitive to be measured and quantified reproducibly in patient blood or other peripheral or proximal fluid samples (such as CSF) by an assay of choice. These markers should be acutely released into the fluids following injury and show a distinct temporal signal pattern. The identification of a unique TBI biomarker(s) or surrogate brain cell injury markers that meet these criteria would provide physicians with an objective method for early diagnosis of brain injury and enable early assessment of severity, intervention, and monitoring disease progression. MultiplPart of the pathology of CNS injury is characterized by secondary effects, including the inflammatory response to TBI. Cytokines are key mediators in the process of (neuro)inflammation and incrWith their elongated axonal and dendritic processes, neurons are exposed to shear forces associated with the whiplash trauma of a concussion. Acute plasma membrane permeablility, or mechanoporation, compromises cell integrity and is linked to diffuse axonal injury in response to a mechanical impact-73.Tau protein is a member of microtubule-associated proteins involved in maintaining cytoskeletal structure and axonal transport. It is expressed by CNS neurons and oligodendrocytes and found primarily in axons. Traditivide infra), studies have demonstrated degradation of MBP isoforms as a result of TBI[Mylein basic protein (MBP) is released with myelin debris that accumulates with axonal damage in the injured brain or spinal cord. MBP is one of three proteins comprising the myelin sheath essential for axonal impulse conduction. MBP mart of TBI,80.Neuron Specific Enolase (NSE), Microtubule-associated protein 2 (MAP-2) and ubiquitin C-term hydrolase L1 (UCH-L1) all display differential expression patterns in TBI patients. NSE, a glycolytic enzyme isoform of neurons, has been documented to increase following head trauma, but haset al. assessed the long-term release of MAP-2 in blood 6 months post trauma by ELISA immunoassay and found that severe TBI patients had significantly higher serums levels of MAP-2 compared to normal non-TBI patients. TBI patients in a vegetative state, as assessed by the GCS, however, showed no increase in serum MAP-2 versus controls. This suggests that MAP-2 could provide insight into the mechanism of neuronal remodeling as well as discriminate between patients with deficits in consciousness and increased risk of unfavorable outcomes[Microtubule-associated protein 2 (MAP-2) is a cytoskeletal-associated protein localized to dendrites of neurons that is believed to function in the growth and maturation of dendrites as well as cytoskeletal organization. Previououtcomes.Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been identified in a cell death culture assay and is verified by ELISA to be significantly increased in TBI patients. Neurodeet al. showed both SBDP145 and SBDP120 increased in patients post-TBI, with SBDP145 present immediately post-trauma and SBDP120 most accurately measured 24 hr post-injury. SBDP CSF levels greater than specific thresholds were shown to correlate with poor outcome and mortality and the temporal expression of SBDP for non-surviving patients differed from that of surviving patients. Thus, if cross-reactivity and breakdown specificity is controlled, SBDPs in CSF may aid to predict the severity of injury and mortality[Spectrin breakdown products (SBDPs) have been identified as potential TBI biomarkers in rat CSF fluid. αII-Speortality.Astroglia are the most abundant cells in the human cerebral cortex and respsevere TBI in CSF and serum, relate to poor outcome, and are predictive for mortality[GFAP is an intermediate filament that is highly enriched in CNS astroglia, but is also expressed in Schwann cells and olfactory ensheathing glia of peripheral nerves-112. GFAortality,55,113. ortality,114. Meaortality. GFAP brortality.For a brain cell specific protein to be a trauma marker, either it should be selectively expressed in response to the trauma and then discharged into fluids, or cytosolic proteins released solely from injured neurons and glia with compromised membrane integrity or dying brain cells,117. A sProteomic studies of injured brain or spinal cord tissue are being done in these injury models and are providing lists of protein changes that are difficult to interpret due to the complexity of events at and around a dynamically changing lesion site and variations between models,40,42. Iselection much easier.Proteomic TBI marker projects on biofluids using rodent injury models have been few due to naturally limited available fluid amounts,127, butet al. describes a workflow using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS/MS in conjunction with off-line nano-LC sample fractionation[Hanrieder ionation. In theiionation,138. Labtargeted proteomic screen using a well-characterized in vitro cell-based trauma model as a starting point for TBI marker candidate identification[et al., submitted; manuscript in preparation). A suitable selection strategy needs to be applied to any trauma-release protein list to eliminate proteins found in healthy human plasma and to focus on brain-specific proteins[Due to the confounding complexity of clinical TBI and clinic-resembling animal injury models, we propose a fication-144. Thification. We are fication . This maybe due to as variables such as the patient’s varying blood–brain barrier integrity, hemorrhage, brain cell protein leakage, as well as waves of brain cell death. This is unlike healthy CSF or plasma with constant and physiologically controlled protein levels allowing for sample preparation with reproducible protein amounts. These ide novo generation, lengthening the assay development time. Thus, the lack of multiplex capacity may exclude using the ELISA platform as initial validation tool of candidate TBI markers in patient samples[The accepted "gold standard" of single-protein measurements is the ELISA immunoassay, which takes advantage of the specificity and diversity of IgG antigen recognition. Yet, while ELISA is well touted for its high sensitivity (~1 pg/mL), it is not without limitations. ELISA m samples.By not relying on antibody-antigen binding, quantitative mass spectrometry is well suited to meet the challenge of overcoming the verification bottleneck where immunoassays cannot be applied. MRM-MS is quickly becoming the preferred method of candidate biomarker verification because of the discriminating power of mass analyzers to accurately measure and quantify multiple specific proteins within a single sample set. Specific peptide fragments (via trypsin digestion) corresponding to the candidate proteins are selected to act as stoichiometric representatives (or surrogates) within a complex patient CSF or blood sample. The mass spectrometer  is then set to scan for the precursor peptide ion, fragment the precursor in the collision cell, and then select for a specific precursor fragment (known as a transition). Because the mass spectrometer is not expending resources scanning through all the ions within a complex patient sample, the signal from less abundant peptides are no longer being masked by highly abundant ions, partially addressing the problems with high dynamic range limitations. Additionally, MRM provides a more cost-effective alternative for quantification compared to traditional ELISA methods by using stable isotope-labeled internal standards of the selective candidate peptides. Using the method of isotope dilution, isotopiThe MRM-MS platform is ideally suited to address the challenge of validating several marker candidates at once (multiplexing) and measuring their levels together with candidate TBI markers reported in the literature. This is in large part due to advances in in pre-analysis enrichment methods as well Combining a targeted screen, a focused selection strategy, and a stepwise approach from preclinical validation towards clinical translation offers a feasible pipeline for candidate TBI marker identification and preparation for its diagnostic use. Validation through a stepwise increasing sample cohort and moving from severe TBI CSF to matching plasma samples and then to mTBI plasma samples will provide verification where experimental analyses and patient samples are matched with appropriate positive controls along the way.Moreover, the emergence of targeted MS-technologies brings promise to the development of an efficient biomarker discovery to verification pipeline for TBI. This pipeline could consist of the initial application of proteomics technologies in the form of comparative 2D-PAGE and shotgun LC-MS/MS to identify and discover candidate biomarkers from trauma and healthy subject samples. This is followed by the development of quantitative MRM-MS to assess the biological significance of these markers followed by clinical validation in a larger scale. With the possibility of multiplexing using proteomic methods such as MRM-MS, the time required for preclinical verification can be reduced as tens of marker candidate proteins can be monitored concurrently in clinical samples. This process will help narrow the pool of potential surrogates from which the most specific and easily measured candidates can be chosen for clinical validation and assay development.The authors declare that they have no competing interests.SS, RROL, IBW, and JAL reviewed the relevant literature. All authors participated in the drafting of the manuscript, and all have read and approved the final manuscript.


In particular, due to rapidly decreasing costs and its suitability for identifying the genetic determinants of a phenotypic trait of interest, whole genome sequencing is expected to be one of the major driving forces in industrial biotechnology in the coming years. We present some of the recent studies that have successfully applied high-throughput sequencing technologies for finding the underlying molecular mechanisms for (a) improved carbon source utilization, (b) increased product formation, and (c) stress tolerance. We also discuss the strengths and weaknesses of different strategies for mapping industrially relevant genotype-to-phenotype links including exploiting natural diversity in natural isolates or crosses between isolates, classical mutagenesis and evolutionary engineering.The application of genome-scale technologies, both experimental and  Modern strain design process starts with the identification of a set of genetic target modifications anticipated to improve yield, productivity, robustness or other performance relevant features. Subsequently, the whole arsenal of analytical techniques available today to characterize the transcriptome, metabolome, proteome and other -omes of production strains helps to identify opportunities for additional rounds of metabolic engineering. However, capabilities for strain design and construction have dramatically expanded due to advances in DNA synthesis and sequencing, moving from narrow studies focused on a few genes to broad and deep searches that seek to optimize traits at the genome-scale level  carbon source utilization, (b) product formation, and (c) stress tolerance.Escherichia coli to efficiently utilize glycerol, a possible alternative carbon feedstock derived from biodiesel processes  in the same gene. With the exception of GSU0514, a putative transcriptional regulator, no mutations in other genes were detected, demonstrating that a single base-pair change resulting in a non-synonymous change in amino acid can markedly influence the metabolic phenotype of G. sulfurreducens. The authors verified their hypothesis by introducing the single-base-pair mutation into the wild type and monitoring the growth on lactate.Summers and colleagues  and CEN.PK113-7D, a preferred laboratory strain for industrial biotechnology research. Considering only metabolic genes the authors detected a total of 85 metabolism-specific non-synonymous SNPs distributed across 158 metabolic genes with clear correlations between physiology and metabolic pathway enrichment. Specifically the non-synonymous SNP enrichment in GAL1 and GAL10 was correlated with the lower galactose uptake rate of S288c compared to CEN.PK113-7D suggesting obvious targets for improving galactose respiro-fermentative metabolism in S288c. In a parallel study, Nijkamp et al.,  are responsible for higher ergosterol concentration in CEN.PK. The specific biochemical mechanisms were linked to alterations in protein structure with direct implications in the stability and substrate affinity of the proteins by sequence-based analysis of the nsSNPs effects, comparative modeling of protein structures, free energy differences and stability analysis of native and mutant proteins, and scanning of binding pockets and functional residues. The authors utilized the detected metabolic SNPs for constructing a production platform of triterpenoids, which resulted to a S. cerevisiae strain capable of producing 500% more β-amyrin than the control strain.Madsen E. coli as demonstrated by the study of Andersen et al.  and compared it with the genome of an industrial enzyme-producing A. niger strain (CBS 513.88). The comparison of the two strains revealed an exceptionally high number of SNPs per kilobase, several of which were accumulated in metabolic pathways essential to protein synthesis and acid production. In connection to protein production the proline, aspartate, asparagine, tryptophan and histidine biosynthesis pathways were enriched in mutations. Mutations relevant for the production of citric acid were found in the TCA cycle, electron transport chain, plasma membrane-bound ATPase and the GABA shunt.Comparative genomics strategies for identifying genetic basis of increased product formation can be extended to less well-studied industrial hosts than yeast or et al.,  and resulted in an isobutanol yield equal to 76% of the theoretical maximum.Smith et al.,  strain N315. The method led to identification of a S. clavuligerus strain that produces a known antibiotic, holomycin, which is not produced in detectable quantities by the wild type strain. Genome re-sequencing revealed that the evolved strain had lost pSCL4, a large plasmid constituting 21% of the genome content of S. clavuligerus. In addition S. clavuligerus acquired several SNPs in genes that have been shown to affect secondary metabolite biosynthesis offering a mechanistic explanation to the activation of holomycin production in the evolved strain.In a recent study Charusanti Thermoanaerobacter species was used in combination with flux analysis and microarray experiments to explain not only why the strain X514 produces higher yields of ethanol from xylose compared to the 39E strain but also to explore other significant differences in the physiology of the two strains with respect to ethanol production  and aspartate carbamoyl transferase involved in pyrimidine biosynthesis from carbamoyl phosphate (pyrB) were independently mutated in the two strains. Using homology-based structural modeling the authors suggested that the location of the altered amino acids residues with the protein structures of AdhE suggest possible alterations in co-factor specificity, catalytic efficiency and/or stability although this needs to be tested with further experiments.Starting from a single colony of a wild-type et al.  creating a very large pool of segregants from a cross, (2) selecting a specific segregant population from this pool that is resistant to e.g. chemical stress, and (3) bulk estimation of allele frequencies in this pool by either microarrays or next generation sequencing. When the X-QTL approach was used with 16 different chemical resistance traits including ethanol and oxidative stress (paraquat) tolerance, it identified between 1 and 24 major genetic loci controlling each trait. The study was later extended to 6 pairwise crosses of additional S. cerevisiae strains to identify whether observed the genetic complexity of traits is mainly dependent on the trait or on the genetic background of the parent strains  or minus (negative) ′′ effect as a result of gene inactivation or modification of residues that cause severe alteration in protein functionality, evolution experiments can select for mutations with more subtle effects providing opportunities for finding novel functions or functional domains in genes and proteins. Additionally, the temporal order in which mutations arise can be easily established in evolutionary engineering studies by sequencing isolates or populations at different time points. This information can be correlated with temporal order of phenotypic changes allowing establishing a putative causative role of specific mutations as well as epistatic relationships between mutations.Evolutionary engineering uses a selection pressure and allows the microorganism to evolve naturally in a chemostat, through sequential batch propagation, or in plate propagation, and thus it relies on the inherent capacity of the cell to introduce adaptive mutations Sauer, . Fast geClassical genetics in the form of crosses and QTL analysis has not been used extensively in microbial cell factory engineering applications both due to lack of familiarity with genetics methods among strain engineers and also due to limited resolution of traditional QTL mapping techniques. However, modern mapping methods like X-QTL are significantly faster than classical methods, and coupled with next generation sequencing have also potential for base pair-level resolution. One of the major benefits in crossing approaches is that the starting genetic variation is controlled as the parental strains can be genotyped, and mature statistical genetics tools can be used to identify and prioritize major variants. Crosses are also the only comprehensive and unbiased way to map the phenotypic effects of genetic changes between two strains that differ at significant numbers of loci. The major drawback is that the approach is limited to sexually reproducing microbes and in particular to microbes where haploid progeny from a cross can be easily isolated like yeast. Crosses can of course also only sample limited genetic variability, as the mutations have to be present in one of the parental strains.Despite impressive progress in methods for mapping the genetic basis of industrially relevant phenotypes, there is still need to speed up the process of finding and verifying genotype-metabolic phenotype links . The proE. coli temperature adaptation study could be constructed in a reasonable time frame.All these developments allow to determining an increasing number of genetic variants underlying metabolic phenotypes, but do not help in verifying the causative effects of these variants or selecting a subset of them for practical use in production strains. Ideally all combinations of high confidence mutations linked to phenotypes of interest discovered in the genotyping studies would be tested in clean wild type backgrounds using high throughput genetic modification techniques such as multiplex automated genome engineering (MAGE) or other recombineering approaches (Wang et al., E. coli more predictive models of e.g. metabolic networks can be used as a scaffold for interpreting and prioritizing genotyping data (Lewis et al., With these challenges in mind, bioinformatics methods similar to those that are being employed increasingly in modern next generation sequencing-based human genetics can also contribute significantly in prioritizing genetic changes that need to be experimentally tested. These methods include enhanced approaches for predicting effects of mutations protein structure or expression based on sequence or structural features (Francesconi et al., Impressively rapid developments in sequencing technologies have revolutionized fields like human genetics and epigenetic studies. These same developments can also help to revolutionize microbial cell factory engineering by providing a more solid and comprehensive genetic basis for engineering efforts. However, sequencing alone will only provide the starting point for cellular engineering and significant advances in synthetic biology techniques, bioinformatics and modeling, as well as high-throughput phenotypic screening will be needed to realize the potential of increased sequence information.


Sauropods are often imagined to have held their heads high atop necks that ascended in a sweeping curve that was formed either intrinsically because of the shape of their vertebrae, or behaviorally by lifting the head, or both. Their necks are also popularly depicted in life with poses suggesting avian flexibility. The grounds for such interpretations are examined in terms of vertebral osteology, inferences about missing soft tissues, intervertebral flexibility, and behavior. Osteologically, the pronounced opisthocoely and conformal central and zygapophyseal articular surfaces strongly constrain the reconstruction of the cervical vertebral column. The sauropod cervico-dorsal vertebral column is essentially straight, in contrast to the curvature exhibited in those extant vertebrates that naturally hold their heads above rising necks. Regarding flexibility, extant vertebrates with homologous articular geometries preserve a degree of zygapophyseal overlap at the limits of deflection, a constraint that is further restricted by soft tissues. Sauropod necks, if similarly constrained, were capable of sweeping out large feeding surfaces, yet much less capable of retracting the head to explore the enclosed volume in an avian manner. Behaviorally, modern vertebrates generally assume characteristic neck postures which are close to the intrinsic curvature of the undeflected neck. With the exception of some vertebrates that can retract their heads to balance above their shoulders at rest , the undeflected neck generally predicts the default head height at rest and during locomotion. Sauropod necks were but one remarkable aspect of an altogether remarkable vertebrate. The necks of many sauropod taxa comprised a larger proportion of the presacral axial skeleton than found in any extant non-avian, with individual vertebrae representing extremes of pneumaticity, elongation, and size. The combination of relatively tiny head, elongate neck, and enormous body has posed fascinating questions regarding sauropods, including how they fed, moved, and simply how they appeared in life.Since only their fossilized bones remain, and usually incomplete and imperfectly preserved at that, even their skeletal reconstructions have been subject to differences in interpretation and sometimes artistic liberties. The overall bauplan of these great giants remains controversial (see below), let alone how they might have held their heads and used their necks in life. Settling the essential questions of sauropod feeding habits and the role of their remarkable necks in feeding will be challenging, since the origin and role of the long neck of the giraffe remains controversial despite their being available for direct observation, as living and behaving animals. Even if alive today, some contention could be expected regarding how the sauropod got its long, long neck. But given only their fossils, much must be inferred and little can be observed directly. This review attempts to summarize what can be concluded about sauropod neck articulation based on correlations between function and  form. The methodology is necessarily inferential and incremental, accumulating a coherent explanation that, while highly incomplete and speculative, is at least consonant with what can be derived from other sources.By analogy, imagine that giraffes were extinct, and known only by their desiccated bones, with some cervical columns remaining in apparently close articulation but in opisthotonic pose Unlike other sciences where the subjects under study are extant and their behaviors  can be observed directly, in paleontology, when the subjects are extinct their behaviors can only be inferred. The process of scientific inference regarding extinct behavior is therefore indirect and conjectural, and many of the presumptions about how these animals lived out their lives rely on tacit intuition and the selection of modern examples that appear to support a given conjecture.Since their first discovery, sauropods have long been the subject of speculation, e.g., that they had a sprawling stance Note that the above speculations are suggestions not only about potential function  but about behavior, and unless outright refuted as physically impossible, are subsequently adopted – or not – based on their appeal and support by analogical reasoning (see below). There is also a tendency to propose conjectures that are not scientifically testable  yet seem compelling, popular, and make for a satisfactory story Recently, however, there has been increasing use of a method to challenge the mythology using observations of modern vertebrates and certain bridging assumptions to ‘ground-truth’ proposals about sauropod pose, flexibility, and behavior. The application of this methodology to examine the mythology is the subject of this review.Conjectures about sauropod neck function, physiology, and feeding behavior are invariably based on skeletal reconstructions by illustrations or mounts and while such reconstructions are very familiar and seemingly authoritative, they often amount to hypotheses or conjectures incorporating significant artistic interpretation (see below). Some effort will be devoted to this issue, since sauropod reconstructions, whether physical or pictorial, are often used uncritically, as will be discussed. The relationships between osteological form and biological function in general, and of vertebral articular geometry and joint articulation in particular are becoming increasingly understood, and recent studies are confirming that vertebral osteology can tell us something about pose and flexibility. The vertebrate neck is not merely a chain of bones and joints, but a system, and observable correlates between structure and function in the necks of extant vertebrates are becoming better understood, thus permitting more principled application towards interpreting sauropod neck function.“… the truth will out. Nature's phenomena will agree or they'll disagree with your theory. Although you may gain some temporary fame and excitement, you will not gain a good reputation as a scientist if you haven't tried to be very careful…” There seems a universal tendency to confer greater trust upon simple parsimonious explanations of natural phenomena, those that capture some essence in few words and to hold broadly without exception. It has been suggested that, based on how extant amniotes hold their heads in alert rest, that sauropods assumed a posture with the neck is maximally extended (dorsiflexed) at the base and the head maximally ventriflexed The necessarily speculative nature of theorizing in paleontology is unfortunately susceptible to the social phenomenon of ‘group polarization’, as speculations are adopted and repeated by secondary sources Regarding sauropod necks, for instance, while historically they have long been depicted with a wide range of combinations neck curvature (from straight to sharply reflex-curved) and slope at mid-neck  Analysis of neck curvature in modern vertebrates provides a means to estimate neck curvature in sauropods. The starting point is to distinguish intrinsic curvature as that which remains in a vertebral column when all intervertebral joints are in an undeflected state. Osteological mounts of extant vertebrates are valuable resources illustrating intrinsic curvature and how it arises from their particular osteology. When the vertebral columns of extant birds, reptiles and mammals are assembled with the successive vertebrae spaced according to their separations in life, and with each joint undeflected, the columns assume a familiar and characteristic intrinsic curve associated with that taxon in an osteologically neutral pose (ONP). The process of estimating intrinsic curvature is of course neither absolute nor exact, nor is it immune to artistic bias and measurement error, in particular as regards speculation about the thickness of the intervertebral separation for extinct vertebrates.Despite their common depiction with rising curvature at the base of the neck, reconstructions of the undeflected neck in sauropods in the cervico-dorsal vertebral columns suggest they were straight where the neck transitions into the anterior dorsals To depolarize the dichotomy between sauropod necks as straight-and-stiff-and-horizontal versus curved-and-flexible-and-upright requires replication and independent confirmation, and convergence of contributions from multiple directions. This will allow for more substantive pursuits, such as seeking deeper explanations for their extreme specializations. But proposals about sauropod neck curvature, pose, head height and so forth have been confounded and conflicted in the literature, and progress will likely require that they be understood in basically the following order:intrinsic curvature of the vertebral column in the undeflected state.intervertebral flexibility.habitual pose for a variety of activities, including feeding, locomotion, and alert rest.characteristic motions involved in browsing, drinking, display, surveillance.vertical and lateral reach, feeding envelopes versus reachability volumes.Just as the sauropod neck is becomes better understood in terms of topics (1–3), the post-cervical skeleton is as well, permitting refined estimations of the motions and characteristics of the vertebrate as whole. Importantly, studies of sauropod forelimbs and pectoral girdles Estimates of head elevation as based on osteology alone is generally lower than that suggested by an analysis of forces and moments Inferences about sauropod necks  rapidly lead from the hard evidence, the fossil material, to speculation. Even the reassembly of the undeflected vertebral column requires understanding how they were connected by soft tissues, and yet their intervertebral joints are not known and can only be inferred. Inferences about habitual neck posture and movement are even more derivative – and necessarily more speculative – as they build upon assumptions about intervertebral flexibility, which must build upon assumptions about the intervertebral joints, and so forth.0) for some extinct taxon t0. Since P(t0) cannot be observed directly, an indirect argument is offered that involves an ‘osteological correlate’ O that reliably co-occurs with P in extant taxa and which is reliably preserved in the fossil record. O and P should be “causally associated” The Extant Phylogenetic Bracket (EPB) 0The co-occurrence of O and P is examined for the so-called extant phylogenetic bracket (EBP), namely the set of taxa that comprise the closest-related outgroup to the extinct taxon t0 might be offered as evidence that this taxon also exhibited property P: Given the physical correlate holds for extant taxa, the presence of the physical evidence O in a fossil of an extinct taxon tIf the osteological correlate O is exhibited by all taxa in the EPB, then a so-called ‘Type I’ inference is supported for the extinct taxon, i.e., While some inference about the extinct taxon is well supported by observations on extant counterparts, the inference necessarily remains a speculation – just a “more constrained speculation” An invocation of EPB is particularly weak if, in addition to neglecting osteological correlates, draws conclusions based on only a limited sampling of extant taxa that exhibit the given property: The undeflected state of an intervertebral joint, geometrically defined by centering the associated pre- and postzygapophyses.A vector constructed from cotyle center to condyle center, used to quantify curvature along a vertebral column .The angular difference between successive vertebral axes. Intervertebral curvature is zero when the axes are geometrically collinear .The curvature of a vertebral column in ONP, as distinguished from curvature induced by joint deflection .The set of all achievable combinations of mediolateral and dorsoventral flexion. As applied to a vertebral column: the set of poses , also termed a ‘reachability envelope’.Contact between vertebrae that limits angular deflection at a vertebral joint and provides load-bearing bracing against disarticulation. Osteological stops may be present independently for dorsiflexion and for mediolateral flexion, or not at all.During intervertebral joint flexion, displacement between pre- and postzygapophyses is limited by the surrounding ligamentous synovial capsule, which draws taut prior to their disarticulation, preserving a ‘safety factor’, a minimal overlap . The ZSF provides a not-to-exceed limit on joint deflection, which is further restricted by soft tissues and behaviorally (see below).To support speculation that some extinct taxon had some unpreserved property  based on 1) observation of that property in closely-related living forms, the EPB, and 2) an ‘osteological correlate’. Presence of that correlated physical evidence in the extinct taxon supports inference that the unpreserved property was also present (see below).The first two of the above five tasks concern estimation of intrinsic curvature and the extremes of what the joint geometry might allow – basic geometric (kinematic) aspects of sauropod neck shape and flexibility, and do not concern estimating their habitual poses, postural preferences, or behavioral tendencies. The success with which neck curvature and flexibility is replicated through the manipulation of the dry bones of extant vertebrates might be used to gauge the feasibility of estimating sauropod neck curvature and flexibility Quantification of intrinsic curvature and joint flexibility requires first establishing the undeflected state of the intervertebral joints. The osteologically neutral pose or ONP A vertebral column in ONP reveals the characteristic curve of the undeflected neck, which provides an important guide to how that neck is utilized Intervertebral joint flexion, in general, involves one vertebra rotating about an instantaneous or ‘momentary’ center of rotation relative to the other Wedge-shaped intervertebral disks contribute to the intrinsic lordotic or kyphotic spinal curvature of amphiplatyan vertebral columns, such as mammalian vertebrae and particularly apparent in the human lumbar spine Numerous articulated sauropod cervical vertebral columns have been found with their central condyles deeply inserted within cotyles . Since both the central articulation and the zygapophyses are diarthrodial, with free sliding motion within their capsules, angular deflection at the centrum results in predominantly a translation or gliding motion of parallel articular surfaces, which is especially apparent as the postzygapophyses sliding across prezygapophyses. That translation must be arrested at some point otherwise disarticulation will occur. It is expected that sauropods, like modern vertebrates, arrested motion prior to disarticulation, preserving a residual overlap or zygapophyseal safety factor (ZSF).Intervertebral joints flex dorsoventrally, mediolaterally, and in combination  to define a range of motion (ROM). Manipulation of dissections of turkey cervical columns A conservative ZSF of 0.5 was used to estimate the relative neck flexibility in two diplodocids In some vertebrates, in addition to limiting deflection by the ligamentous synovial capsule surrounding the zygapophyses, intervertebral joint flection may be limited by physical contact between vertebrae, e.g., between the postzygapophyses of one vertebra against the neural spine of the more caudal vertebra It has been suggested that dorsiflexion in sauropods was also limited by osteological stops, given their presence in Camelus bactrianus, Giraffa Camelopardalis and Struthio camelus While some extant cervical vertebrae are braced ontologically as they reach the limits of dorsal or lateral deflection, many extant vertebrates do not exhibit apparent morphological adaptations (pers. obs.). Nor does osteological bracing against excessive dorsiflexion appear present in sauropods . Each successive pair of vertebral axes  defines The normal division of human spine into regions of intrinsically lordotic or kyphotic spinal curvature arises partly by wedge-shaped intervertebral disks as mentioned, and partly by the vertebral osteology . The osteological contribution can be subtle but accumulative, as in the slight wedge shape of the vertebrae in the lumbar spine Many birds also have ventral OIC cranially, which together with the rise at the base of the neck, creates a sigmoid curve with an inflection in curvature mid-neck . ReptileCaution is needed to distinguish between behaviorally-induced curvature and that which is intrinsic to the osteology, particularly when attempting to draw broad generalizations about default behavioral postures Speculation regarding the relationship between ONP and characteristic poses of the neck during rest and locomotion for sauropods seems of less importance to understanding their biology than how they used their necks for feeding. The relationship between ONP and the characteristic pose for feeding in modern herbivores is straightforward only for grazers, while many low browsers will take advantage of vegetation that requires raising the head above ONP, and high browsers will very frequently feed by ventriflexion far below ONP Given the rarity and inaccessibility of physical mounts of dinosaur skeletons, skeletal drawings and composites have traditionally used to reconstruct and subsequently analyze sauropod vertebral columns. More recently, 3D digital models are being used in preference to relying on 2D artwork Skeletal illustrations have been relied upon to both summarize the bauplan of a given taxon of sauropod, and as source material on which to base estimates of head height and speculations regarding feeding, and so forth. Significant artistic liberties are sometimes noted The macronarian Camarasaurus, for example, is usually depicted to have had a sharply rising neck at the base, largely due to illustrations based on the juvenile C. lentus CM 11338. The original specimen was preserved in a severe opisthotonic posture, with the cervicals wrenched back and the zygapophyses displaced out of articulation . The skeWhile skeletal reconstructions may incorporate artistic liberties, some degree of independent verification is afforded by the detailed steel engravings or photographs of the individual vertebrae illustrations were published in the original descriptions by C. Gilmore, J.B. Hatcher, W. Janensch, W. Wiman and others. These illustrations can be scanned, composited, and placed into articulation to roughly reconstruct their ONP  [31]–[32The neck of the iconic Giraffatitan brancai mount  is reconWhile extreme in the case of the Berlin mount, it is not uncommon for neural spines to be restored in sauropod skeletal mounts as if they were in ONP. The Apatosaurus ajax at the Yale Peabody Museum (YPM VP 001980), for instance, has a gently-curved sigmoidal-shaped neck. Close inspection shows that the zygapophyses are centered, as if the vertebrae were in ONP. Still closer inspection, requiring a ladder to reach up and tap on the darkly-varnished plaster, reveals an artistic amalgam of real material and plaster (pers. obs.). The gracefully-ascending curve to the neck appears to have been conceived first, then the details of the restoration made to neatly fit that vision.The neck of Camarasaurus lentus USNM 13786-310D  was presThe Denver Museum of Nature and Science Diplodocus longus DMNS 1494 is also mounted with an upward bend in the neck at its base . Again, 2D skeletal illustrations provide only limited insight into vertebral articulation, but have been resorted to given limited access to, and manipulation of, original specimens, especially those that are mounted. Yet entire articulated vertebral columns can be manipulated in a virtual 3D space provided their morphology is converted to digital form. Fossil vertebrae can be digitized to capture their surface morphology and subsequent digital retrodeformation An alternative to digitization of original material is to begin with a set of deformable 3D models can be subsequently formed to closely resemble the morphology of the original specimens. Deformable 3D surfaces can be created using subdivision surface model techniques Through a laborious process of building then adjusting generic models of axial and appendicular elements to fit specimens, eventually entire articulated digital skeletons can be constructed , 22, 23 The following general inferences regarding sauropod vertebral joints appear supported by the EPB, with avia and reptilia as outgroups:Intervertebral central articulations were diarthrotic, with close intervertebral separations.Anterior cervical vertebrae were essentially straight (negligible OIC in ONP).Posterior cervical vertebra had slight ventral OIC in ONPIntervertebral joint flexibility was limited by synovial capsules surrounding the zygapophyses which draw taut prior to permitting disarticulation, preserving a ZSF.Cervical vertebrae were limited in dorsiflexion by the ZSF, not by osteological stops.Specific conclusions regarding sauropod necks would include the need to revise the reconstructions of brachiosaurids and camarasaurids based on 1–3, above. Regarding 4 and 5, above, the relatively ‘stiff’ necks of sauropods  and their kinematics suggest a coherent role for the sauropods neck with regard to feeding. While not yet explored in detail, the following provides a review that draws on the deep understanding of neck function in extant vertebrates towards better understanding the corresponding function of sauropod necks. As discussed below, some of the insight derives not by analogy with birds, but by how the analogy with birds fails, and yet resembles that of another, very distant group: browsing mammalian herbivores.While the relatively inflexibility of sauropod necks compared to birds is sometimes viewed with skepticism The cervical column may be regarded kinematically as a spline-like chain of fixed-length links between joints of limited angular mediolateral and dorsoventral flexibility Consider the consequences of varying vertebral length, count, and flexibility, either singly or in combination. First, increasing vertebral length alone increases reach linearly and the surface area of the reachability envelope quadratically. Increasing intervertebral flexibility, particularly caudally, also increases surface area roughly linearly . Next, while holding overall neck length constant, increasing vertebral count while trading off intervertebral flexibility and vertebral length off can produces a tradeoff, resulting in the same reachability envelope. But increasing vertebral count without proportionately reducing intervertebral flexibility greatly dramatically increases the kinematic redundancy of the neck A substantial literature on extant amniotes  shows that:Taylor, Wedel, and Naish “living animals do not habitually maintain their necks in ONP. Instead …C0: “the neck is maximally extended at the cervico-dorsal junctionC1: “and maximally flexed at the cranial-cervical junctionC2: “so that the mid-cervical region is near vertical.C3: “This is true even in apparently short-necked animals. …C4: “The fact that elevated, extended necks are widespread across Amniota means thatC5: “elevated necks should be assumed for sauropods in the absence of evidence to the contrary.C6: Elevated neck postures for sauropods are indicated by the extant phylogenetic brackets at the levels of Saurischia, Archiosauria, Diapsida, Reptilia, and Amniota.C7: “0 if a physical property O is identified, that is correlated with P in all members of the extant outgroups comprising the EPB, and O is present in traces of the extinct taxon: Recall that the EPB method supports a Type I inference about an unpreserved property P in some extinct taxon tRegarding the conjectured C0–C4 The behavioral claims C0–C4 While some mammals can and do rest in an alert state by retracting the head to balance it atop a subvertical column, the far more widely-adopted posture in quadrupeds is to cantilever the head and neck before the shoulders, in approximate ONP. The weight of the head and neck is then supported passively by means of suspension through some combination of dorsal musculature and ligaments in tension The osseous labyrinth containing the semicircular canals constitutes a potential osteological correlate for supporting inferences about the preferred or stereotypic head posture in extinct vertebrates The habitual orientation of the head relative to gravity is a behavior property, one that is fortunately correlated with the gravitational orientation of the LSC, and hence of potential use as an osteological correlate An EPB-supported inference of the gravitational orientation of the cranium in the extinct vertebrate could be inferred from 1) the observed orientation of the LSC within the cranium of an extinct vertebrate Proceeding caudally through the occiput , the gravitational slope of the neck at the atlas-axis is constrained as well. Three independent lines of evidence can thus be combined. The LSC data supports a postulated slope for the atlas-axis relative to horizontal, and post-cervical skeletal reconstructions suggest the gravitational slope of the anteriormost dorsal vertebrate , and in the middle, ONP studies of re-articulated cervical columns in the undeflected state, suggest the relative slopes at their two ends. The three lines of evidence combine satisfactorily with the following caveats :Sauropod heads were held in alert rest with a relatively small inclination α of the LSC.Sauropod cranio-cervico joints are held undeflected in alert rest.Sauropod necks are suspended, with intervertebral joints in approximate ONP .Sauropod cervico-dorsal vertebrae are held in approximately ONP in alert rest.So progressing from the cranium through the cervical column caudally and into the cervico-dorsal transition, a consistent  global picture is emerging. But speculations about how sauropods held their head in alert rest, when not otherwise occupied, has perhaps less relevance to sauropod biology compared to how the animal used its neck for feeding, and secondarily, while engaged in locomotion.Starting with the bare bones, plus caveats about their intervertebral separations based on modern vertebrates with similar articulations, the cervical vertebral columns of sauropods, relieved of their opisthotonic pose , are revSauropod skeletal reconstructions indicate a range of slopes for the anterior dorsal columns. Variation in the resting height and gravitational orientation of the head can be attributed primarily to variations in body plan without postulating any mechanism  for creating an upward bend in the base of the neck. Thus despite having no intrinsic upward bend at the base of the neck ONP, the sauropod head could have been placed at a substantial elevation above the shoulders, or at or even below the shoulders, simply due to the slope of the anterior dorsal column. Even modest dorsiflexion at the base could then produce several meters of additional head elevation in those sauropods with especially long necks, and those with long necks and high resting height could also ventriflex to bring the head down to browse low (as well as drink water). The once-held distinction between low versus high browsers is not sharply defined.Upper bounds on neck mobility are predicted geometrically for extant vertebrates, and those criteria, applied to sauropod necks, predict less-than-avian flexibility, presuming sauropod necks did not disarticulate (more than once per lifetime). Extant vertebrates that do not have a sigmoid curve to the neck, sauropod necks were well-suited for directing the head to different locations on a ‘feeding envelope’ surface rather than to any point within the volume within that surface (think cow not swan). While intervertebral flexibility was comparable to most that of reptiles, and less than most birds, they more than made up for ‘stiff’ necks by their absolute length. While some sauropods literally went to extraordinary lengths to sweep out a ‘feeding surface’ swath in front of them by flexing their necks at full extension dorsoventrally and mediolaterally, their necks were neither pre-curved avian-style nor sufficiently flexible to fully explore the volume of space contained within that surface. Despite some uvam acerbam arguments that sauropods held their heads high  sauropod necks were incapable of the prerequisite ability to retract the head sufficiently to balance it's weight above the shoulders, adopted passive suspension of the head, like extant vertebrates that share this inability. But how sauropods held their head when inactive seems of lesser importance to understanding their feeding behavior .Several of the conclusions in this review seem negative, about what sauropod necks did not look like, and what they did not do, and which popularizations are not scientifically supported and should be abandoned. For instance, none were shaped like swan necks, and there is no support for the persistent suggestion they held their heads high habitually. Perhaps the most useful such negative is that sauropods were not unique – at least, there is no evidence to suggest that what is known about the articulation, suspension, and function of extant archosaur vertebral columns does not apply as well to the sauropods, despite their extremes.All is certainly not negative: there are many EPB-supported (or supportable) hypotheses to propose and to test, given the commonality between extinct and extant archosaurs – inferences which investigators have barely begun to be explore. The lateral semicircular canal evidence is compelling (and likely to become more so), as is the upper-bounds on flexibility implied by vertebrates sensibly preserving a safety factor of overlap at the zygapophyses. Osteological bracing (both its presence and absence) and its relationship to loads imposed upon necks at the limit of flexibility has only been noted in a few cases. The kinematic importance of a sigmoidal intrinsic curve to the neck has been well appreciated for birds, and that extends to extinct vertebrates that share a sigmoidal design. But the implications of a neck that is a simple monotonic arc, devoid of a built-in inflection point, has not been previously explored either in extant vertebrates or in sauropods, and yet clearly has relevance to browse-gathering efficiency and behavior. Preconceived notions and ill-supported presuppositions will be replaced increasingly by newly-conceived notions as methodology replaces mythology in the study of sauropods.The 3D models that appear in this review have been developed in Autodesk Maya Regarding the modeling of individual bones, conventional digital modeling employes two somewhat disparate choices: importing a polygonal mesh of vertices that form a piecewise planar approximation to a surface from sampled positions across the given object which, given sufficiently many samples, creates an apparently smooth replica of an actual specimens  that represent sufficient morphology to capture the major osteological features  sufficient to model a range of variation across taxa for appendicular elements, and across both taxa and position within a vertebral column for axial elements. To model a specific dorsal vertebral column, for example, a generic dorsal vertebra model is duplicated multiple times to represent the first, mid, and last vertebrae of a given specimen. Each instance is then individually sculpted to match the shape and dimensions of its original counterpart, based on archival material, photographs, and, when available, digitized surface scans, CT, or other point-sampled data of actual specimens. Vertebral osteology in archosaurs varies sufficiently smoothly that missing elements can be interpolated over small intervals. Interpolation of missing or severely damaged elements based on adjacent elements is justifiably criticized as being somewhat speculative, as in the case of the restoration of C13–C15 in the Carnegie Museum specimen Apatosaurus louisae CM 3018 Next, blend shape animation The skeleton is then rigged to become fully articulated , the digital joints can be exercised to explore the intervertebral range of motion along the axial skeleton, reachability envelopes, and so forth, as exemplified by the figures in this review.Figure S1(TIF)Click here for additional data file.Figure S2(TIF)Click here for additional data file.File S1(ZIP)Click here for additional data file.Movie S1(MP4)Click here for additional data file.Movie S2(MP4)Click here for additional data file.Movie S3(MP4)Click here for additional data file.Movie S4(MP4)Click here for additional data file.Movie S5(MP4)Click here for additional data file.Movie S6(MP4)Click here for additional data file.Movie S7(MP4)Click here for additional data file.Movie S8A turntable movie depicting the spinal cord (red) entering the foramen magnum of Kaatedocus siberi.(MP4)Click here for additional data file.Movie S9Animation of Camarasaurus from its death pose into a life pose near ONP.(MP4)Click here for additional data file.Movie S10(MP4)Click here for additional data file.Movie S11(MP4)Click here for additional data file.Movie S12(MP4)Click here for additional data file.


Trigonisca ameliae) preserved in ‘Anthropocene’ Colombian copal, dated to ‘post-Bomb’ and 10,612±62 cal yr BP, respectively, and obtained sequence reads using the GS Junior 454 System. Read numbers were low, but were significantly higher for DNA extracts prepared from crushed insects compared with extracts obtained by a non-destructive method. The younger specimen yielded sequence reads up to 535 nucleotides in length, but searches of these sequences against the nucleotide database revealed very few significant matches. None of these hits was to stingless bees though one read of 97 nucleotides aligned with two non-contiguous segments of the mitochondrial cytochrome oxidase subunit I gene of the East Asia bumblebee Bombus hypocrita. The most significant hit was for 452 nucleotides of a 470-nucleotide read that aligned with part of the genome of the root-nodulating bacterium Bradyrhizobium japonicum. The other significant hits were to proteobacteria and an actinomycete. Searches directed specifically at Apidae nucleotide sequences only gave short and insignificant alignments. All of the reads from the older specimen appeared to be artefacts. We were therefore unable to obtain any convincing evidence for the preservation of ancient DNA in either of the two copal inclusions that we studied, and conclude that DNA is not preserved in this type of material. Our results raise further doubts about claims of DNA extraction from fossil insects in amber, many millions of years older than copal.Insects preserved in copal, the sub-fossilized resin precursor of amber, have potential value in molecular ecological studies of recently-extinct species and of extant species that have never been collected as living specimens. The objective of the work reported in this paper was therefore to determine if ancient DNA is present in insects preserved in copal. We prepared DNA libraries from two stingless bees (Apidae: Meliponini:  Labidura herculeana, which appears to have become extinct during the mid-twentieth century as a result of habitat destruction and the introduction of non-indigenous predators by humans Nymphalis polychloros now considered extinct in Britain but present in Europe Museum entomology collections represent an unparalleled wealth of species biodiversity and distribution data for all insect groups from around the globe. This information is becoming increasingly accessible as more museums host searchable catalogues online. Such archived samples are irreplaceable, hence destructive sampling for attempted DNA extraction is usually discouraged by museum curators. Although there has been only limited success in obtaining DNA sequences from samples such as detached legs from air-dried insects Another consideration is the difficulty in collecting insects in certain regions, due to funding constraints, armed conflict, local wildlife conservation laws, etc. It is not unheard of for entomologists to receive prison sentences for collecting insects without the appropriate permits Similar information might be obtained from sub-fossil insects in copal, the sub-fossilized resin precursor of amber, renowned for preserving insects with life-like fidelity The objective of the work reported in this paper was to determine if ancient DNA (aDNA) is present in insects preserved in copal. Intuitively, one might imagine that the complete and rapid engulfment in resin, resulting in almost instantaneous demise, might promote the preservation of DNA in a resin entombed insect. Within this protective environment, DNA preservation might be better than in an air-dried museum specimen. This rationale has been used to explain the remarkable preservation of DNA in million-year-old insects present in amber inclusions -2 for ×5 min, with 180° rotation between the two exposures) before use. Aqueous solutions we similarly irradiated for 15 min. Personnel wore protective clothing including forensic suits, face masks, hair nets, goggles and two pairs of sterile gloves at all times. Specimen preparation and DNA extraction were carried out in a Class II biological safety cabinet in one laboratory within the facility, and library preparation in a second, physically-isolated laboratory. Modern insect DNA had never been handled in the building within which these laboratories are located.All manipulations up to and including preparation of sequencing libraries were carried out within the aDNA facility at the University of Manchester, which comprises a suite of independent, physically isolated laboratories, each with an ultrafiltered air supply maintaining positive displacement pressure and a managed access system. All surfaces within the laboratories were periodically sterilised by UV irradiation and cleaned with 30% bleach and 70% ethanol, and all utensils and equipment were treated with DNA-Away (Molecular Bioproducts) before and after use. Items such as test tubes were UV irradiated  for 20 h at 55°C with gentle agitation Each insect was removed from the dissolved copal and rinsed in 2 ml absolute alcohol followed by 2 ml UV-irradiated HPLC grade water. DNA extraction was immediately carried out. For non-destructive DNA extraction, the insect was incubated in 1 ml extraction buffer . The non-destructive and destructive extracts for each specimen were barcoded and pooled and two libraries prepared, one for each specimen. The GS FLX Rapid Library MID Adaptors Kit was used in accordance with the manufacturer's instructions, except that the DNA fragmentation step was omitted, due to the anticipated short lengths of any aDNA molecules in the extracts. The library was eluted in 30 µl TE buffer  and this entire amount used for emulsion amplification with the Titanium emPCR kit, again following the manufacturer's instructions, including the specific recommendations for short-length amplicon libraries. As enrichment was very low, three emulsions were prepared for each library and these combined before loading on to the sequencing chip. Sequencing was carried out with the Titanium sequencing kit using the preset filters, except for changing the vfTrimBackMinimumLength parameter from 84 flows to 20 flows so that short reads would not be excluded. All the test reads passed the standard quality controls and control reads were of good quality. As the test read numbers were very low, duplicates were not removed prior to downstream processing. Sequence reads were converted into FASTA format and searched using MEGAN4 As we have previously reported –6, two hits were obtained for the sequences from the non-destructive extract, and eight for the destructive library , gave sequences, but other than one read which partially aligned with a bumble bee mitochondrial DNA sequence, none of these had convincing matches with taxa related to stingless bees.Mycobacterium tuberculosis in human bone We do not believe that our negative results, from two sequencing libraries prepared from four extracts, can be ascribed entirely to technical incompetence. In previous and current projects we have successfully prepared aDNA libraries for next generation sequencing from –6, which aligned with sequences from bumble bee (one read) and common environmental bacterial taxa (nine reads), others gave lower significance scores that might be interpreted as poor quality aDNA sequences. For example, one 61-nucleotide read obtained from the non-destructive library could be interpreted as 23 nucleotides of bee DNA (hit to Apis florea) followed by 21 nucleotides of insect gut bacterium . Similar chimeric interpretations could be made for other reads, as many of these contained at least one short segment that aligned with an Apidae sequence. However, in most cases, the Apidae alignment was less than 20 bp, and few were longer than 30 bp, which is hardly the basis for a convincing argument bearing in mind the overall lengths of the sequence reads.With the older specimen the absence of aDNA is incontrovertible because the only reads that we obtained were artefacts. For the younger inclusion it might be possible to argue that the reads derived from highly degraded aDNA from the insect and its microflora at the time of death. Although the vast majority of the reads gave very low significance scores in BLAST searches against the complete nucleotide database as well as a database made up of Apidae sequences, a case could be made for some of these being insect or microfloral in origin. As well as the few reads that gave BLAST matches with a E value <10Our conclusion that DNA is not preserved in insect inclusions in copal contrasts with the reports of successful DNA amplification from dried and pinned museum specimens DNA degradation is influenced by factors such as oxygen and water content, ambient temperature and time since death of the organism Theoretical and empirical data indicate that aDNA fragments might be present in well preserved geological material up to at least 100,000 years in age, and suggest that some material up to one million years might yield sequence data Table S1Sequence reads.(DOC)Click here for additional data file.


Somatic Genome Variations (SGV) are referred to as intercellular variability of genomes in somatic tissues of the same organism. These can manifest as single-nucleotide changes, short DNA sequence changes (<1kb), short tandem repeat variations, retrotransposition of mobile genome elements (i.e. SINE and LINE), copy number variations and subchromosomal structural abnormalities , structural chromosome abnormalities observed at microscopic level (>2-5 Mb), alterations to chromosome morphology (i.e. fragile sites), aneuploidy (gain/loss of whole chromosomes), polyploidy (gain of haploid chromosome sets). Several lines of evidences have been reported that SGV can play a role in human biodiversity and disease. For instance, it is generally recognized that somatic genome changes produced by genomic and chromosomal instability are cancer-causing. However, related phenomena are rarely addressed in non-malignant tissues. Current genomics essentially uses technologies which operate with DNA isolated from a large pool of cells and analyzes interindividual genomic variations, whereas single-cell genomic approaches are much more rarely applied. This probably explains why SGV are significantly less appreciated. Nevertheless, previous biomedical research does provide initial data that (i) SGV appear to be widespread in human cell populations; (ii) intercellular genomic diversity seems to be associated with a number of neurological, psychiatric and immune diseases, chromosomal syndromes and cancers as well as appear to be involved in critical biological processes ; (iii) molecular cytogenetics does provide technical solutions for studying single-cell genome variations at molecular resolutions. Therefore, a need appears to exist for additional attention to an underappreciated area of single-cell genomics aimed at studying SGV.Current Genomics (Hot Topic Issue on SGV) is to gather the knowledge about causes and consequences of SGV by addressing the experience of leading experts in fields of human genetics, genomics and molecular cytogenetics. This attempt appears to be successful, inasmuch as the line of reviews has provided for an integrated view of how SGV can be involved in human interindividual diversity, normal prenatal development, aging and pathological changes associated with a number of diseases. The issue begins with theoretical considerations about possible phenotypic effects of SGV and about inevitable changes of current concepts in genomics (epigenomics) resulting from research of SGV. Then, a brief overview of SGV in health and disease is given. The issue continues with two articles dedicated to SGV during human prenatal development. The first one describes recent data on intercellular genomic changes in early human embryos and suggests possible effects at further prenatal developmental stages. The second one gives a timely overview of SGV in extra-embryonic tissues and provides convincing evidence that these do play a significant role in the normal placentation. The next article presents an original hypothesis suggesting one of the most common genetic abnormalities in human newborns (trisomy 21) to be a result of intercellular genomic variations in fetal tissues. Furthermore, an extensive overview of SGV manifesting as aneuploidy involving chromosome 21, which are associated with a broad array of diseases, is given. To end the description of SGV in human embryonic and fetal tissues, a review of ontogenetic genome variations is provided. Surveying data on intercellular genome variability from conception to late ontogeny, it was possible to show that SGV are involved in controlling cell numbers during development and aging. Additionally, a phylogenetic model of “dynamic genome” was adapted to cell populations suggesting similar genetic processes to take part as during phylogeny as during ontogeny. Further, the origin of genetic mosaicism produced by SGV manifesting as copy number variations (one of the most common type of genomic variations) is described. According to authors’ data and to the literature, this type of SGV is likely to from during embryonic development remaining stable (cell proportions) as long as 20 years. Continuing evaluations of SGV in liveborns, a review of mosaic small supernumerary marker chromosomes, which represent a frequent type of chromosome abnormalities, is presented and the importance of such cases for prenatal diagnosis is underlined. Diagnostic problems related to SGV and possible ways of their solutions are further described in the next review. Here, an overview of genomic and chromosomal instabilities as well as literature data on identification of SGV has allowed to come to an optimistic conclusion that it is possible to propose recommendations on molecular cytogenetic diagnosis and clinical interpretation of SGV. Focusing on medical aspects, it would be interesting to evaluate SGV in a certain disease. This task was successfully completed by a review addressing genomic instabilities in schizophrenia. Finally, a review showing the potential of modeling SGV (somatic copy number variation) and germline genomic variation for biomedical research is presented.The intention of this special issue of All together, the articles in this Hot Topic Issue provide an exciting review of current SGV research that can stimulate readers to pay more attention to single-cell and somatic cell genomics forming a basis for further studies in this area of genomics and epigenomics.This special issue of Current Genomics is dedicated to the memory of our close relative and colleague, Ilia V Soloviev, a talented young researcher and a pioneer of molecular cytogenetics, genome and chromosome research, whose prodigious work has formed our current research directions.


DNA double-strand breaks are among the most deleterious lesions induced by ionising radiation. A range of inter-connected cellular response mechanisms has evolved to enable their efficient repair and thus protect the cell from the harmful consequences of un- or mis-repaired breaks which may include early effects such as cell killing and associated acute toxicities and late effects such as cancer. A number of studies suggest that the induction and repair of double-strand breaks may not always occur linearly with ionising radiation dose. Here we have aimed to identify and discuss some of the biological and methodological factors that can potentially modify the shape of the dose response curve obtained for these endpoints using the most common assays for double-strand breaks, pulsed-field gel electrophoresis and microscopic scoring of radiation-induced foci. For many years, our knowledge of both the health effects of and the molecular and cellular responses to ionising radiation exposure has been limited to the high dose range, above 100 mSv, due to a lack of sufficiently large and well controlled cohorts for epidemiological studies on one hand, and a lack of experimental tools for assessing low dose responses on the other hand. Based on the available evidence, a linear no-threshold model was generally assumed for cancer risk. Over thow doses.Chromosomal DNA is the most important cellular target damaged by exposure to ionising radiation. Radiation-induced DNA lesions include abasic sites, oxidated bases and sugars, strand breaks and cross-links within or between the complementary DNA strands or between DNA and surrounding proteins. Importantly, radiation causes clusters of such lesions along the track of the ionising particle,8. It isStrand breaks are among the most highly studied DNA lesions induced by ionising radiation. This is partly because of their important contribution to the toxic, mutagenic, clastogenic and carcinogenic effects of radiation, but may also be explained by the availability of a wide range of detection and quantification methods for these particular lesions.2O2 which shows a strong quadratic response at high concentrations[Ionising radiation-induced DNA strand breaks form following attack of the sugar phosphate backbone either by direct DNA radical production or by radicals formed through water radiolysis in the vicinity of the DNA (indirect effect). DSB form when two such nicks are present in opposite DNA strands within one or two helical turns. They seem to result mainly from the attack of multiple radical hits rather than the transfer of one radical between strands. Howevertrations due to ttrations.The ratio of SSB to DSB yields produced by sparsely ionising radiation is commonly estimated to be on the order of 25–40, based on the detection of relaxed circular vs. linear plasmid DNA in agarose gels following irradiation of supercoiled circular plasmids. A recent study, however, which has utilised a direct end-labelling approach for SSB detection, suggests that the true SSB yield may be 10 fold higher than previously assumed. The inaUp until about a decade ago it was impossible to study DSB induction at radiation doses of relevance in occupational or radiodiagnostic settings, due to the very limited sensitivity of the DSB detection methods available, such as neutral filter elution or pulsed-field gel electrophoresis (PFGE), which all measure DSB indirectly through the associated decrease in average molecular weight or length of chromosomal DNA and in general require doses of at least several gray to detect any significant effect, though a detection limit of less than 1 Gy was reported by one group for an optimised PFGE-based assay.Over the past 15 years, radiation-induced foci (RIF), each representing hundreds to thousands of individual proteins involved in the DNA damage response which accumulate in the vicinity of a DSB,22, haveAlthough initial DSB induction by ionising radiation can conceptually be expected to occur linearly with dose, there are number of factors that can affect DSB measurements by any of the above assays. FigureFor PFGE-based assays, mechanical shearing and nuclease attack can contribute to DNA fragmentation that is independent of radiation exposure. This ‘noise’ limits the dose range available for investigation. Excessive DNA degradation can be minimised, but not eliminated, by embedding cells into low gelling temperature agarose, incubation of samples in chelating agents to inhibit nucleases prior to lysis and strict avoidance of any risk of contamination of samples with DNAse.Measurement of the fraction of DNA released into the gel reveals a sigmoid dose response and requires accurate molecular weight analysis of fragment distributions and calibration with I-125 for absolute quantification of DSBs. The useReplication forks can cause DNA molecules to be trapped in the agarose matrix, resulting in reduced mobility and associated underestimation of DNA breakage. Caution should therefore be used when interpreting PFGE results obtained with proliferating cells containing a significant S phase fraction . Careful optimisation of staining conditions and close monitoring of the antibody performance are required in order to obtain consistent results. Staining artefacts can typically be distinguished from ‘true’ foci based on their different morphology. Subtle differences can, however, be lost in maximum projection images, so ‘live’ imaging of slides may be preferable.Absolute yields of DSB per unit radiation dose change linearly with the DNA content, as observed for foci frequencies in cell lines with different DNA content. Also, cScoring of foci relies on setting threshold criteria for foci size, signal intensity and overall morphology of spots to distinguish ‘true’ foci from gamma-H2AX ‘speckles’ (which may form at the sites of transcription ‘bubbles’), antibody aggregates and non-specific binding of the antibody to other targets. Co-localisation of gamma-H2AX foci with 53BP1 is generally assumed to reflect true DSB,42 and dAs foci form as a biological response to DNA damage, a period of at least several minutes post exposure is required before they can be microscopically detected; the exact minimum delay depends on the individual signal to noise level which varies between cell types and is also affected by experimental protocols and reagents used for foci immunostaining. ConsequSecondary gamma-H2AX foci have been reported in irradiated and bystander cells in association with replication stallage-47 and tCurrent automated and manual foci scoring methods tend to underestimate RIF yields at high damage levels, thereby causing a deviation from the linear dose response relationship towards saturation. Using the highest possible optical resolution, manual, rather than automated scoring and scoring of gamma-H2AX rather than 53BP1 foci at high damage levels  and a slow component (a few hours) to be calculated. The bipDose-independent, biexponential kinetics are also widely observed using foci assays, but typically with longer half times; e.g. Horn et al. reportedNeumaier et al. reportedIn conclusion, the seemingly simple task to determine the shape of the dose response curve for DSB induction and repair is, at closer inspection, associated with numerous technical and conceptual caveats and uncertainties that should be considered when interpreting any experimental data. New assays have considerably advanced our understanding of the way cells respond to radiation-induced damage. It is becoming increasingly clear that multiple biological processes, but also methodological factors, may cause the dose response to deviate from linearity. Estimating their impact on the effect of radiation at a tissue or organism level remains a major challenge. In this context extrapolation of results from single experimental studies to draw conclusions on the most appropriate dose–response model to use for the protection of populations against the health effects of ionising radiation may be seen as unwise and potentially misleading.The authors declare that they have no competing interests.SB and KR carried out the initial literature review and drafted the manuscript. SDB contributed to the design and helped to draft the manuscript. All authors read and approved the final manuscript.


In 2010 we edited a Frontiers Special Topic on “An update on neurocognitive impairment in schizophrenia and depression” . In the first of three articles, Hoffman and Hampson review recent work on functional connectivity in AVH and suggest that the core mechanism for AVH involves a more complex functional loop rather than a single impaired pathway. Implicated regions include Wernicke's area, its right homolog, putamen, and left inferior frontal cortex. They also propose a number of important recommendations for future studies.Diederen et al. provide a review of voice hearing in non-psychotic individuals, an important group to study to understand the underlying mechanisms of AVH in the absence of other psychiatric symptoms and medication. One observation they make is that individuals prone to hallucination may have an enhanced bias to auditory stimulation localized to the anterior cingulate regions. They also found evidence that decreased cerebral dominance, often found in schizophrenia, may not be directly related to AVH, again underscoring the importance of studying this population.Along similar lines, Larøi discusses how AVH in patients differs from AVH in non-patients. He highlights several important aspects of these studies including the present of highly negative emotional context in the patient AVHs as well as the early onset of AVHs in the non-patients. Some key suggestions for future research are proposed as well.Nygård et al. focus on differences between patients and controls in functional connectivity across wide-spread networks identified with independent component analysis (ICA) including the default mode network and the so-called task-positive network located in regions subserving executive function. Their findings revealed differences in the degree to which patients up-regulated the default mode network and also a deficit in the down regulation of the anterior default mode network. The study of the dynamic interplay between different functionally connected networks is an emerging area and will undoubtedly play an important role in illuminating brain dysfunction in complex mental illnesses such as schizophrenia.Repovš and Barch studies functional connectivity in the context of multiple working memory loads and patients, siblings, and healthy controls. They also evaluated the relationship among different networks as well as within networks. Interesting observations related to increases in some pairs of regions, e.g., default mode network and fronto-parietal connectivity while decreases in other pairs, e.g., cingulo-opercular and cerebellar at the lowest load level whereas the connectivity between the first pair was modulated by load. The second pair showed consistent differences in patients and their siblings and supports the view that altered functional connectivity in schizophrenia is a stable characteristic.The final paper tackles the difficult problem of using imaging data to classify patients and controls. A unique aspect of this study is Du studies a wide number of intrinsic networks during both extended rest and task during an auditory oddball task. Using a novel analysis approach she obtains impressive results which suggest that despite the great interest in resting fMRI data for its ease of use and ability of patients to perform, a functional task which is relatively easy to perform may provide utility above and beyond rest fMRI in certain cases. Another key finding in this study is that classification information, though not the same across all networks, is wide-spread across the brain, and by incorporating multiple networks one can significantly improve performance.In summary, the current collection of articles represents a wide range of topics related to cognitive impairment in schizophrenia and incorporates new approaches related to intrinsic networks identified at rest or during a task. The current selection of articles also shows how recent developments in structural and functional neuroimaging, may further advance our understanding of this devastating disorder. The more complete picture of these disorders provided by the study of functional and structural connectivity, combined with carefully evaluated tasks, and resting data will likely be important tools in future diagnostics and treatment evaluation.


Gastric antral vascular ectasia is a not so well-understood, and more rare, gastrointestinal manifestation of Systemic Sclerosis which can lead to chronic anemia. A high suspicion and better understanding of this rare manifestation is needed for early detection and treatment. Therapeutic regiments include iron supplementation with acid suppressive therapy, while endoscopic intervention has been shown to be successful in most cases, with gastrectomy or antrectomy rarely needed. Gastric antral vascular ectasia (GAVE) was first defined in 1984 by Jabbari et al. . GAVE is a rare manifestation of systemic sclerosis. A recent large retrospective study of 264 patients with SSc found a prevalence of 5.7% . Eighty In some studies GAVE has been shown to be more prevalent in lcSSc while in other studies in dcSSc patients , 7, 8. PAn association between rapidly progressive skin changes and the development of early onset GAVE  may also exist. A subset of 16 patients from the series of 28 patients in the article by Ingraham et al. were diagnosed with early-onset GAVE. Nine out of these 16 patients also had rapid progression of their cutaneous disease  . CeribelThe autoantibody profile may also give clues to the risk of developing GAVE, and a speckled pattern appears to carry a higher risk . A recenThe pathogenesis of GAVE is not fully understood though it has been noted in the past when pathology specimens from GAVE patients were examined that there is a loose attachment of the distal gastric mucosa to the underlying muscularis externa, where in the setting of dysmotility the antral mucosa may prolapse through the pylorus , 3, 10. GAVE is considered one of the manifestations of the spectrum of vascular alterations in systemic sclerosis . StudiesMost patients with lcSSc and GAVE present with iron deficiency anemia or symptoms attributable to iron deficiency anemia, such as weakness, severe fatigue, or dyspnea on exertion. Thus a high index of suspicion is often needed in order to make the diagnosis in a timely fashion. Patients with dcSSc have a tendency to present with occult blood in the stool, and occasionally one may present with overt gastrointestinal bleeding though melena or hematemesis is unusual , 10. RecThe appearance of GAVE is longitudinal red stripes or rugal folds in the antrum, radiating out in pattern resembling spokes from the pylorus. Alternatively, multiple cherry-red spots may be seen, but this mosaic pattern is far less common. Antral biopsies typically show capillary dilation with focal intravascular thrombi and fibromuscular hyperplasia in the lamina propria . Management of GAVE ranges from conservative symptomatic therapy to endoscopic interventions to surgery. The mainstays of symptomatic therapy and medical management are iron supplementation, proton pump inhibitors, and blood transfusion if anemia is severe and symptomatic . There aEarly diagnosis of GAVE in SSc patients is important because endoscopic management can successfully treat this. It is important to educate primary care physicians who take care of these patients to be aware of slight drops in the hemoglobin of SSc patients and to refer such patients for endoscopic evaluation. While lcSSc patients typically have a good outcome after local therapy, dcSSc are more difficult to treat, with a need for recurrent electrocoagulation and blood transfusions and an increased risk for renal crisis.Several unique endoscopic interventions have been reported to be successful in the management of GAVE. One popular technique is argon plasma coagulation, which has been shown to be effective in short- and medium-term followup in maintaining a rise in hemoglobin and giving transfusion independence to formerly transfusion-dependent patients within 2-3 treatment sessions . OccasioAnother endoscopic technique is neodymium-yttrium aluminum garnet (Nd:YAG) laser photocoagulation. A study of 45 patients treated with Nd:YAG laser reported that, at an average of two-year followup, normal hemoglobin levels were seen in 87% of all patients and transfusion independence was found in 24 of 28 initially transfusion-dependent patients . There have been case reports describing many other successful therapies such as endoscopic band ligation , endosco There have been reports of success in two small series of patients using intravenous cyclophosphamide to treat systemic sclerosis-associated GAVE or remission of GAVE when cyclophosphamide is used to treat other serious disease manifestations such as pulmonary disease , 20. Gastrectomy or antrectomy is the only cure for GAVE, but it is rarely needed in the modern era with such a vast array of medical and endoscopic therapies available. Gastric antral vascular ectasia continues to be a not well-understood manifestation of SSc patients, but an increase awareness and early diagnosis can improve outcome since local therapy can be successful. There is a need for a noninvasive therapy, though currently the best approach in the care of these patients is close followup and repeated local therapy.