Investigation of Genetic Defects in Severe Combined Immunodeficiency Patients from Turkey by Targeted Sequencing.

Primary immunodeficiencies (PIDs) represent a large group of disorders with an increased susceptibility to infections. Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiencies (PIDs) with marked T-cell lymphopenia. Investigation of the genetic aetiology using classical Sanger sequencing is associated with considerable diagnostic delay. We here established a custom-designed, next-generation sequencing (NGS)-based panel to efficiently identify disease-causing genetic defects in PID patients and applied this method in SCID patients of Turkish origin with previously undefined genetic aetiology. We used HaloPlex enrichment technology, a targeted, NGS-based method which was designed to diagnose patients with SCID and other PIDs. Our HaloPlex panel included a total of 356 PID-related genes, and we searched disease-causing mutations in 19 Turkish SCID patients without a genetic diagnosis. The coverage of targeted regions ranged from 97.47% to 99.62% with an average of 98.31% for all patients. All known SCID genes were covered with a percentage of at least 97.3%. We made a genetic diagnosis in six of 19 (33%) patients, including four novel disease-causing mutations identified in RAG1, JAK3 and IL2RG, respectively. We showed that this NGS-based method can provide rapid genetic diagnosis for patients suffering from SCID, potentially facilitating clinical treatment decisions.

Identifying potential predictors of pain-related disability in Turkish patients with chronic temporomandibular disorder pain.

BACKGROUND: The aims of this study were to examine whether patients' psychosocial profiles influence the location of pain, and to identify the clinical and psychosocial predictors of high levels of pain-related disability in temporomandibular disorders (TMD) patients with chronic pain at least 6 months in duration. METHODS: The Research Diagnostic Criteria of TMD (RDC/TMD) data for Axis I and II were obtained for 104 consecutive patients seeking treatment.Data were analyzed using descriptive statistics, t-test, Mann-Whitney U-test, chi-square test, One-way ANOVA, Kruskal-Wallis test, and binary multiple logistic regression tests. Patients were classified into two groups according to Graded Chronic Pain Scale scores: Grade III and IV were scored for patients with high levels of pain-related disability, whereas Grade I and II were scored for patients with low disability. RESULTS: Muscle and joint pain were found in 64.9% and 31.8% of the patients, respectively, and 27.3% of the patients suffered from both muscle and joint pain.Psychosocial disability was found in 26% of patients. There were no statistically significant differences among the diagnostic subgroups with regards to the demographic, behavioral, psychological, and psychosocial characteristics. Patients with high levels of pain-related disability had significantly higher depression, somatization, pain intensity and jaw disability scores than those with low levels of pain-related disability.Patients with high levels of pain-related disability were more likely to have higher pain intensity, to report higher somatization symptoms and functional impairment, and were less likely to have joint pain than those with low levels of pain related disability. CONCLUSION: In conclusion, the Turkish version RDC/TMD, based on a dual axis system, may be used to screen chronic TMD patients at high-risk for pain-related disability who need comprehensive care treatment program.

Anharmonicity, mode-coupling and entropy in a fluctuating native protein.

We develop a general framework for the analysis of residue fluctuations that simultaneously incorporates anharmonicity and mode-coupling in a unified formalism. We show that both deviations from the Gaussian model are important for modeling the multidimensional energy landscape of the protein Crambin (1EJG) in the vicinity of its native state. The effect of anharmonicity and mode-coupling on the fluctuational entropy is in the order of a few percent.

Regulation of lymphoid-specific immunoglobulin mu heavy chain gene enhancer by ETS-domain proteins.

The enhancer for the immunoglobulin mu heavy chain gene (IgH) activates a heterologous gene at the pre-B cell stage of B lymphocyte differentiation. A lymphoid-specific element, microB, is necessary for enhancer function in pre-B cells. A microB binding protein is encoded by the PU.1/Spi-1 proto-oncogene. Another sequence element, microA, was identified in the mu enhancer that binds the product of the ets-1 proto-oncogene. The microA motif was required for microB-dependent enhancer activity, which suggests that a minimal B cell-specific enhancer is composed of both the PU.1 and Ets-1 binding sites. Co-expression of both PU.1 and Ets-1 in nonlymphoid cells trans-activated reporter plasmids that contained the minimal mu enhancer. These results implicate two members of the Ets family in the activation of IgH gene expression.

ETS-core binding factor: a common composite motif in antigen receptor gene enhancers.

A tripartite domain of the murine immunoglobulin mu heavy-chain enhancer contains the muA and muB elements that bind ETS proteins and the muE3 element that binds leucine zipper-containing basic helix-loop-helix (bHLH-zip) factors. Analysis of the corresponding region of the human mu enhancer revealed high conservation of the muA and muB motifs but a striking absence of the muE3 element. Instead of bHLH-zip proteins, we found that the human enhancer bound core binding factor (CBF) between the muA and mu elements; CBF binding was shown to be a common feature of both murine and human enhancers. Furthermore, mutant enhancers that bound prototypic bHLH-zip proteins but not CBF did not activate transcription in B cells, and conversely, CBF transactivated the murine enhancer in nonlymphoid cells. Taking these data together with the earlier analysis of T-cell-specific enhancers, we propose that ETS-CBF is a common composite element in the regulation of antigen receptor genes. In addition, these studies identify the first B-cell target of CBF, a protein that has been implicated in the development of childhood pre-B-cell leukemias.

Transcriptional activation by ETS and leucine zipper-containing basic helix-loop-helix proteins.

The immunoglobulin mu heavy-chain gene enhancer contains closely juxtaposed binding sites for ETS and leucine zipper-containing basic helix-loop-helix (bHLH-zip) proteins. To understand the mu enhancer function, we have investigated transcription activation by the combination of ETS and bHLH-zip proteins. The bHLH-zip protein TFE3, but not USF, cooperated with the ETS domain proteins PU.1 and Ets-1 to activate a tripartite domain of this enhancer. Deletion mutants were used to identify the domains of the proteins involved. Both TFE3 and USF enhanced Ets-1 DNA binding in vitro by relieving the influence of an autoinhibitory domain in Ets-1 by direct protein-protein associations. Several regions of Ets-1 were found to be necessary, whereas the bHLH-zip domain was sufficient for this effect. Our studies define novel interactions between ETS and bHLH-zip proteins that may regulate combinatorial transcription activation by these protein families.

Collective motions in HIV-1 reverse transcriptase: examination of flexibility and enzyme function.

In order to study the inferences of structure for mechanism, the collective motions of the retroviral reverse transcriptase HIV-1 RT (RT) are examined using the Gaussian network model (GNM) of proteins. This model is particularly suitable for elucidating the global dynamic characteristics of large proteins such as the presently investigated heterodimeric RT comprising a total of 982 residues. Local packing density and coordination order of amino acid residues is inspected by the GNM to determine the type and range of motions, both at the residue level and on a global scale, such as the correlated movements of entire subdomains. Of the two subunits, p66 and p51, forming the RT, only p66 has a DNA-binding cleft and a functional polymerase active site. This difference in the structure of the two subunits is shown here to be reflected in their dynamic characteristics: only p66 has the potential to undergo large-scale cooperative motions in the heterodimer, while p51 is essentially rigid. Taken together, the global motion of the RT heterodimer is comprised of movements of the p66 thumb subdomain perpendicular to those of the p66 fingers, accompanied by anticorrelated fluctuations of the RNase H domain and p51 thumb, thus providing information about the details of one processivity mechanism. A few clusters of residues, generally distant in sequence but close in space, are identified in the p66 palm and connection subdomains, which form the hinge-bending regions that control the highly concerted motion of the subdomains. These regions include the catalytically active site and the non-nucleoside inhibitor binding pocket of p66 polymerase, as well as sites whose mutations have been shown to impair enzyme activity. It is easily conceivable that this hinge region, indicated by GNM analysis to play a critical role in modulating the global motion, is locked into an inactive conformation upon binding of an inhibitor. Comparative analysis of the dynamic characteristics of the unliganded and liganded dimers indicates severe repression of the mobility of the p66 thumb in RT's global mode, upon binding of non-nucleoside inhibitors.

Identification of kinetically hot residues in proteins.

A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed.

Relationships between amino acid sequence and backbone torsion angle preferences.

Statistical averages and correlations for backbone torsion angles of chymotrypsin inhibitor 2 are calculated by using the Rotational Isomeric States model of chain statistics. Statistical weights of torsional states of phipsi pairs, needed for the statistics of the full chain, are obtained in two different ways: 1) by using knowledge-based pairwise dependent phipsi energy maps from Protein Data Bank (PDB) and 2) by collecting torsion angle data from a large number of random coil configurations of an all-atom protein model with volume exclusion. Results obtained by using PDB data show strong correlations between adjacent torsion angle pairs belonging to both the same and different residues. These correlations favor the choice of the native-state torsion angles, and they are strongly context dependent, determined by the specific amino acid sequence of the protein. Excluded volume or steric clashes, only, do not introduce context-dependent phipsi correlations into the chain that would affect the choice of native-state torsional angles.

[Primary pathological changes in cells infected with tick-borne encephalitis virus]. / Pervichnye patologicheskie izmeneniia v kletkakh, zarazhennykh virusom kleshchevogo éntsefalita

Synthesis and the content of RNA and protein in cells of the pig embryo kidneys infected with tick--borne encephalitis virus were studied by means of autoradiography and cytophotometry. Acceleration of synthesis and accumulation of RNA (in cellular structures) were estimated as over 125--200% of the initial level followed by a decrease in 12 hr when the virus release from the cells occurred. By the 2nd and 6--8th hr of infection (in the period of synthesis of early and late virus material), two peaks of protein contents over 123--131% were observed. The RNA and protein shifts are well correlated with the virus reproduction stages.

[Ultrastructural changes in central nervous system cells due to arboviruses]. / Ul'trastrukturnye izmeneniia v kletkakh tsentral'noi nervnoi sistemy, vyzyvaemye arbovirusami.

The paper systematizes the data of the literature on ultrastructural changes in the central nervous system (CNS) of experimental animals infected with arboviruses of the Togaviridae family. The main site of virus reproduction in the CNS was found to be nerve cells in which the ultrastructural lesions typical for this group of viruses developed. The main features of these lesions consist in degeneration of rough membranes, ribosomes, and polysomes of the cell, hyperplasia and hypertrophy of smooth membranes, formation of various vacuoles and vesicles in the cell hyaloplasm. Mature virions accumulate in cisterns and cavities of the endoplasmic reticulum, lamellar complex, in vacuoles and vesicles. Each of arboviruses produces in the cells not strictly specific but typical ultrastructural lesions. In the CNS the viruses spread hematogenically, in intercellular and perivascular spaces and dendrites of the nerve cells.

[Ultrastructural changes in the central nervous system of monkeys in poliomyelitis]. / Ul'trastrukturnye izmeneniia v tsentral'noi nervnoi sisteme obez'ian pri poliomielite

The ultrastructure of cells of the central nervous system of Rhesus monkeys in experimental poliomyelitis caused by virus of poliomyelitis Type 3 was studied. It was found out that the virus of poliomyelitis was localized in nerve cells, astocytes, oligodendrocytes, the endothelium of capillaries and macrophages. This justifies the conclusion that pathological changes in the cells referred to the above result from reproduction of the virus therein. Ultrastructural changes in infected cells are divided into three stages: the first stage corresponded to eclipse-phase of infection; the second -- to the period of reproduction of the virus and composition of viral particles; and the third -- to the period following the escape of the virus from the cell. A definite combination of nonspecific changes in organoids of various cells with specific viral inclusions makes the ultrastructural picture of cells in poliomyelitis sufficiently characteristic for differential diagnosis. Pathogenesis of the developing lesions is discussed.

[Morphological diagnosis of intrauterine chlamydia infection and its clinico-anatomical characteristics]. / Morfologicheskaia diagnostika vnutriutrobnogo khlamidioza i ego kliniko-anatomicheskaia kharakteristika.

Clinicomorphological characteristics of intrauterine chlamydiasis (IUC) are given. The authors describe a cottonwool-like meningochorioiditis the observation of which at necropsy allows IUC suspection. Chlamydial etiology of this condition is confirmed by means of electron microscopy and immunofluorescence. Fibroblast proliferation of the pia mater followed by intensive fibrillogenesis plays a most important role in morphogenesis of cottonwool-like nodules. The occurrence of IUC at the necropsies varies depending on the age of foetuses and newborns from 6.2 to 38.7%.

[Ultrastructural pathology of the lymph nodes in tick-borne encephalitis]. / Ul'trastrukturnaia patologiia limfaticheskikh uzlov pri kleshchevom éntsefalite.

Lymph nodes of humans and experimental animals with tick-borne encephalitis (TBE) were studied using electron microscopy. TBE virus reproduction occurred in macrophages, reticular and plasma cells. Severe organelle destruction was found in the above cells and lymphocytes. The results suggest that macrophage, reticular and plasmatic cell death is due to the virus reproduction, while lymphocyte death is due to the toxic effect. It is evident that the outcome is determined by pathological process in lymphoid organs.

[Ultrastructural changes in the central nervous system of mice in tick-borne encephalitis]. / Ul'trastrukturnye izmeneniia v tsentral'noi nervnoi sisteme myshei pri kleshchevom entsefalite.

Tick-borne encephalitis virus is localized in nerve cells and intracellular spaces. According to the nature of ultrastructural changes, the pathological process in nerve cells may be divided into three stages connected with the stages of virus reproduction. In the first stage, the protein-synthesizing systems of the cell are degraded. In the second stage specific virus inclusions form in the cell cytoplasm as aggregates of mature and immature virions alongside with destruction of ultrastructures and growth of smooth membranes. In the third stage degradation of neurons is accompanied by necrobiosis and discomplexation of the surrounding glial elements, processes, cells of the inflammatory infiltrate and capillary walls. A certain combination of nonspecific changes in organelles of various cells with viral inclusions makes the ultrastructural picture of the focus of lesions in tick-borne encephalitis sufficiently characteristic for differential pathological diagnosis.

[Central nervous system lesions in fetuses and newborns in intrauterine infection caused by respiratory viruses]. / Porazheniia tsentral'noi nervnoi sistemy plodov i novorozhdennykh pri vnutriutrobnoi infektsii, vyzvannoi respiratornymi virusami.

Congenital respiratory viral infection is followed in the CNS of fetuses and newborns by grave dyscirculatory disturbances with glia proliferation and nervous tissue edema. The process is mainly localized in the periventricular regions of the brain ventricles. Neurologic and morphologic consequences of these damages in the CNS of fetuses and newborns need further studies.

[Ultrastructural pathology of congenital infection provoked by respiratory viruses]. / Ul'trastrukturnaia patologiia vrozhdennoi infektsii, vyzvannoi respiratornymi virusami.

The ultrastructural study of fetal and newborn lungs in the intrauterine infection caused by respiratory viruses showed the virus reproduction to take place in type II alveolocytes with destructive changes in cell organelles. Each type of virus results in a certain picture of ultrastructural changes. Progressing cell organelles destruction with an intensive cytoplasm vacuolization and virion presence in the cytoplasmic matrix and on the endoplasmic network membranes is characteristic for influenza. Severe cytoplasm vacuolization with lipid formation, homogenization and condensation of membranous bodies, nuclear pyknosis are observed in parainfluenza. RS-infection is characterized by virion formation on the cytoplasmic cell membrane, numerous deformed membranous bodies, filling intercellular spaces and alveolar lumen with rough granular electron-dense material, massive release of organelles into the alveolar lumen. Edema and vacuolization of endothelium, edema and focal disappearance of basal membrane are observed in the capillary walls.

[Pathomorphology of uveitis caused by an echovirus]. / Patomorfologiia uveita, vyzvannogo virusom ECHO.

The monkey eyes infested by inoculation into the eye anterior chamber of the ECHO-19 virus from children with uveitis were studied morphologically. It was established electron microscopically that the inoculated virus is replicated in the capillary endothelium and stromal cells of the uveal tract. Crystal-like accumulation of virions and degenerative changes in the organelles are observed in these cells. Acute granulomatous alterative-exudative-hemorrhagic uveitis with the destruction of the iris, ciliary body and processes and secondary changes in the cornea and crystalline lens with the symptoms of keratitis and cataract develop in the anterior part of the eye as observed in the light microscope. The anterior and posterior goniosynechia develop. An acute inflammatory process is further transformed into a progressing autoimmune inflammation.

[Ultrastructural changes in the CNS of monkeys with the chronic form of tick-borne encephalitis]. / Ul'trastrukturnye izmeneniia v TsNS obez'ian pri khronicheskoi forme kleshchevogo éntsefalita.

The central nervous system of monkeys with chronic tick-borne encephalitis (TBE) shows the following ultrastructural alterations which differ from those in acute TBE: widespread destructive changes in neurons, astrocytes, oligodendrocytes, processes, myelin, vascular walls, severe edema of brain tissue with the signs of a so-called spongious degeneration and the absence of cell proliferation and perivascular cell infiltrations, glial nodules as well as circulatory disorders (hyperemia, stasis, hemorrhages). Few virions with or without and altered supercapsid membrane are detected in the neuronal cytoplasm or in the intercellular spaces; the signs of active virus reproduction in the cells are lacking. Pathologic process is of a degenerative but not of an inflammatory nature; the latter is typical for acute TBE.