Tönnis and the Novel IHDI Radiographic Classification Systems for the Developmental Dysplasia of The Hip (DDH): Evaluation of 406 hips with DDH.

In this study, we aimed to compare the efficiency of Tönnis and the novel International Hip Dysplasia Institute (IHDI) in decision making and in presuming the outcomes in children who had undergone closed reduction and casting. 406 hips of 298 patients who had undergone closed reduction and spica casting were included in this retrospective study. All hips were classified according to Tönnis and IHDI systems. Bucholz-Ogden classification was used for avascular necrosis. The outcomes of patients for each classification system were compared, in terms of the presence of avascular necrosis, redislocations and secondary surgeries at the end of the follow-up period. 318 hips were evaluated as Tönnis grade 2 dysplasia. 24 had avascular necrosis, 9 had redislocations. 79 hips were evaluated as Tönnis grade 3 dysplasia. 18 had AVN, 7 had redislocations. 9 hips were evaluated as Tönnis grade 4 dysplasia 3 had AVN, 4 had redislocations. 203 patients were evaluated as IHDI grade 2 dysplasia. 7 had AVN, 7 had redislocations.185 patients were assessed as IHDI grade 3 dysplasia. 33 had AVN, 11 had redislocations. 18 patients were evaluated as IHDI grade 4 dysplasia. 5 had AVN, 6 had redislocations. Both Tönnis classification and IHDI classification systems are reliable and efficient systems for evaluating the severity and predicting the success of closed reduction and casting for the treatment of DDH. IHDI classification has certain benefits, such as being a practical classification and a better distribution within the groups.

COMT rs4680 and DRD2 rs6275 variants and their association with YMRS scores in children with early-onset bipolar disorder

Background and objectives: Bipolar disorder (BD) is a clinical status with at least one manic, hypomanic or mixed attacks. Genetic factors take part significantly in early-onset BD (EOBD). Dopamine receptors (DRD) act in neurological mechanisms like motivation, learning, memory, and, control of neuroendocrine signaling. DRD2 receptor has been reported to influence the stability of DRD2 transcript. Catechol-O-Methyl transferase (COMT) inactivates catecholamines and Val158Met variation on COMT has effects on COMT activity. This study aims to explore DRD2 and COMT variants in the clinical development of EOBD.MethodsIn this case-control study, 102 EOBD patients and 168 healthy control subjects were used. DRD2 rs6275 and COMT Val158Met variations were detected by real-time polymerase chain reaction (RT-PCR). Young Mania Rating Scale (YMRS) was utilized to determine the EOBD severity.ResultsFor DRD2 rs6275 and COMT Val158Met polymorphisms, no significant relationship was observed in the genotype and allele frequencies between patient and control groups. Nevertheless, TT genotype carriers of DRD2 rs6275 polymorphism demonstrated significantly increased YMRS scores when compared with CC and CT genotype carriers (p = 0.039). Nevertheless, no significant difference was observed between COMT Val158Met genotypes and YMRS scores.ConclusionsWe suggest that the DRD2 rs6275 TT variant can be associated with symptom severity in children with EOBD and can have a clinical significance in EOBD pathogenesis. However, these results need to be confirmed with larger samples of patient and control groups. (AU)

Point-of-care Lung Ultrasound, Lung CT and NEWS to Predict Adverse Outcomes and Mortality in COVID-19 Associated Pneumonia.

Introduction: The appraisal of disease severity and prediction of adverse outcomes using risk stratification tools at early disease stages is crucial to diminish mortality from coronavirus disease 2019 (COVID-19). While lung ultrasound (LUS) as an imaging technique for the diagnosis of lung diseases has recently gained a leading position, data demonstrating that it can predict adverse outcomes related to COVID-19 is scarce. The main aim of this study is therefore to assess the clinical significance of bedside LUS in COVID-19 patients who presented to the emergency department (ED). Methods: Patients with a confirmed diagnosis of SARS-CoV-2 pneumonia admitted to the ED of our hospital between March 2021 and May 2021 and who underwent a 12-zone LUS and a lung computed tomography scan were included prospectively. Logistic regression and Cox proportional hazard models were used to predict adverse events, which was our primary outcome. The secondary outcome was to discover the association of LUS score and computed tomography severity score (CT-SS) with the composite endpoints. Results: We assessed 234 patients [median age 59.0 (46.8-68.0) years; 59.4% M), including 38 (16.2%) in-hospital deaths for any cause related to COVID-19. Higher LUS score and CT-SS was found to be associated with ICU admission, intubation, and mortality. The LUS score predicted mortality risk within each stratum of NEWS. Pairwise analysis demonstrated that after adjusting a base prediction model with LUS score, significantly higher accuracy was observed in predicting both ICU admission (DBA -0.067, P = .011) and in-hospital mortality (DBA -0.086, P = .017). Conclusion: Lung ultrasound can be a practical prediction tool during the course of COVID-19 and can quantify pulmonary involvement in ED settings. It is a powerful predictor of ICU admission, intubation, and mortality and can be used as an alternative for chest computed tomography while monitoring COVID-19-related adverse outcomes.

BDNF Val66Met polymorphism is associated with negative symptoms in early-onset schizophrenia spectrum and other psychotic disorders

Abstract Background and Objectives. To investigate the clinical characteristics of adolescents with early-onset full psychotic disorders either with Brain-derived neurotrophic factor (BDNF) Val66Met (rs6265) or DRD2/ANKK1 Taq1A (rs1800497) polymorphisms.

Acute-phase reactants and cytokines in ischemic stroke: do they have any relationship with short-term mortality?

BACKGROUND: Many unknown risk factors play a role in the etiopathogenesis of stroke. The appearance of inflammatory cells within the damaged tissue after cerebral ischemia suggests that an inflammatory response may play a role in stroke pathogenesis. In our study, we examined whether an association exists between the acute-phase reactants and the levels of cytokines, the volume and diameter of the stroke, and short-term mortality in patients who were diagnosed as acute ischemic a stroke after admission to the Emergency Department. PATIENTS AND METHODS: A total of 50 consecutive patients who applied to the Emergency Service with acute ischemic stroke were enrolled in the study. Their stroke volume were calculated and serum samples were obtained as soon as they arrived into the Emergency Service. The patients were evaluated according to the Glasgow Coma Scale (GCS) and National Institutes of Health Stroke Scale (NIHSS). RESULTS: There was no significant correlations between stroke volume and levels of cytokine and acute-phase reactants in dead patient group or in living patient group. A correlation and statistical significance was found between stroke volume and hospital stay time in living patient group. In addition, GCS and NIHSS scores were correlated with stroke volume and was found a significant statistically. CONCLUSIONS: Scales such as GKS and NIHHS, which evaluate the functional state of patients, are the best indicators for defining prognosis in our daily practices. In addition, we found a positive correlation between levels of CRP (C reactive protein) and prognosis. However, we did not observe a statistically significant correlation between prognosis and other acute-phase reactants such as TNF-alpha, IL-6, IL-8, IL-10, fibrinogen, and leukocytes.

Unusual presentations of scorpion envenomation.

Scorpions are nocturnal arthropods that inject their venom through the victims' skin by stingers. By the envenomation, clinical manifestations in a wide spectrum may occur, including pain at one side and death because of severe cardiopulmonary or neurological abnormalities. Sometimes the victim cannot describe the insect or does not remember even being stung after the event. We present two cases of scorpion envenomation with different and rare clinical situations with a short review of the literature.

An investigation of the anger levels of residents: medical compared with surgical disciplines.

OBJECTIVE: To evaluate medical and surgical residents' anger levels with regard to the department in which they worked, seniority, sex, satisfaction with their work environment, and the number of nightshifts worked per month. The specific situations and persons at whom residents reacted with anger were also investigated. METHODS: 116 randomly selected residents staffed in a university hospital (62 medical and 54 surgical residents) were enrolled. The trait anger and anger expression scale was used to find out the personal anger levels of each participant. The participants also clarified the persons and situations that made them angry at work. RESULTS: Trait anger levels were greater in the surgical residents in their first two years when compared with levels of their senior colleagues (p = 0.033). Mean trait anger levels were greater in the residents who were not satisfied with their department (p = 0.004). Anger levels were not found to be related to the number of shifts per month. Male residents had higher levels of anger than female colleagues (p = 0.019). CONCLUSION: Residents in clinical sciences seem to have the potential to benefit from a screening process in terms of anger and its subcomponents by means of a tool such as the trait anger and anger expression scale during their residency.

Structure of NaeI-DNA complex reveals dual-mode DNA recognition and complete dimer rearrangement.

NaeI, a novel DNA endonuclease, shows topoisomerase and recombinase activities when a Lys residue is substituted for Leu 43. The NaeI-DNA structure demonstrates that each of the two domains of NaeI recognizes one molecule of DNA duplex. DNA recognition induces dramatic rearrangements: narrowing the binding site of the Topo domain 16 A to grip DNA, widening that of the Endo domain 8 A to encircle and bend DNA 45 degrees for cleavage, and completely rebuilding the homodimer interface. The NaeI-DNA structure presents the first example of novel recognition of two copies of one DNA sequence by two different amino acid sequences and two different structural motifs in one polypeptide.

Intrapleural urokinase in the management of parapneumonic empyema: a randomised controlled trial.

We compared the effectiveness of intrapleural urokinase versus normal saline via a thoracostomy tube in the treatment of parapneumonic empyema in a randomised controlled study. Forty-nine patients with parapneumonic empyema were randomly assigned to receive either intrapleural urokinase or normal saline treatment. The daily volume instilled through a chest tube was 100 ml in both groups. Urokinase (100,000 IU/day) was diluted in normal saline before instillation. The mean duration for defervescence was shorter (7 +/- 3 vs 13 +/- 5 days, p<0.01) and the mean volume of drained fluid during the five-day treatment period was significantly greater in the urokinase group (1.8 +/- 1.5 vs 0.8 +/- 0.8 litres, p<0.001) than in the control group. The subsequent decortication rate was 60% and 29.1%, respectively (p<0.001). The duration of hospitalisation was also shorter in the urokinase group (14 +/- 4 vs 21 +/- 4 days, p<0.001) than in the saline group. We conclude that intrapleural instillation of urokinase in the management of parapneumonic empyema provides a better outcome and reduces the need for decortication.

Evidence for mutations that break communication between the Endo and Topo domains in NaeI endonuclease/topoisomerase.

NaeI is a type IIe endonuclease that interacts with two DNA recognition sequences to cleave DNA. One DNA sequence serves as a substrate and the other serves to activate cleavage. NaeI is divided into two domains whose structures parallel the two functionalities recognized in NaeI, endonuclease and topoisomerase. In this study, we report evidence for mutations that break interdomain functional communication in a NaeI-DNA complex. Deletion of the initial 124 amino acids of the N-terminal domain of NaeI converted NaeI to a monomer, consistent with self-association being mediated by the Endo domain. Deletions within a small region of the C-terminal DNA binding domain of NaeI (amino acids 182-192) altered the recognition by NaeI of sequences flanking the NaeI recognition sequence. Substituting Ala for Arg182 within this region had no apparent effect on DNA binding but greatly reduced the extent of DNA cleavage even though it is not part of the catalytic Endo domain. Substituting Ala for Ile185 reduced the extent of DNA binding about 1000-fold. Substituting Ala for Lys189 altered flanking sequence recognition. Residues 182-192 are away from the Endo domain responsible for cleavage and also face away from the modeled DNA binding faces of the apoprotein crystal structure. We propose that residues 182-192 are part of a web that mediates the flow of information between the NaeI Endo and Topo domains.

Crystal structure of NaeI-an evolutionary bridge between DNA endonuclease and topoisomerase.

NAE:I is transformed from DNA endonuclease to DNA topoisomerase and recombinase by a single amino acid substitution. The crystal structure of NAE:I was solved at 2.3 A resolution and shows that NAE:I is a dimeric molecule with two domains per monomer. Each domain contains one potential DNA recognition motif corresponding to either endonuclease or topoisomerase activity. The N-terminal domain core folds like the other type II restriction endonucleases as well as lambda-exonuclease and the DNA repair enzymes MutH and Vsr, implying a common evolutionary origin and catalytic mechanism. The C-terminal domain contains a catabolite activator protein (CAP) motif present in many DNA-binding proteins, including the type IA and type II topoisomerases. Thus, the NAE:I structure implies that DNA processing enzymes evolved from a few common ancestors. NAE:I may be an evolutionary bridge between endonuclease and DNA processing enzymes.

Abasic sites induce triplet-repeat expansion during DNA replication in vitro.

The occurrence of triplet-repeat expansion (TRE) during transmission of genetic information is involved in many neurological and neuromuscular diseases including Fragile X syndrome and myotonic dystrophy. DNA slippage during replicative synthesis appears to cause TRE. The causes of DNA slippage, however, remain mostly unknown. We investigated the effects of abasic sites on the occurrence of TRE during DNA replication in vitro using Escherichia coli Klenow polymerase I as the model polymerase. Here we show that a single abasic site analog, synthesized in the triplet-repeat tract at the 5' end of the template strand, induced dramatic TRE during DNA synthesis. The amount of TRE induced decreased when the abasic site was moved to the middle of the repeat tract, consistent with effectively decreasing the length of the repeat tract. Placing the abasic site in the primer did not induce TRE. TRE was sequence-dependent. The damage-induced increase in growing strand TRE depended on the sequence of the growing strand repeat as AAT approximately ATT > CAG > CTG. The expansions required replication from a primer complementary to the repeat tract. The expanded tracts were sequenced and contained multiple additions of the original repeat. The results imply that DNA damage can play a significant role in generating TRE in vivo.

Effects of temperature, Mg2+ concentration and mismatches on triplet-repeat expansion during DNA replication in vitro.

The human genome contains many simple tandem repeats that are widely dispersed and highly polymorphic. At least one group of simple tandem repeats, the DNA trinucleotide repeats, can dramaticallyexpand in size during transmission from one generation to the next to cause disease by a process known as dynamic mutation. We investigated the ability of trinucleotide repeats AAT and CAG to expand in size during DNA replication using a minimal in vitro system composed of the repeat tract, with and without unique flanking sequences, and DNA polymerase. Varying Mg2+concentration and temperature gave dramatic expansions of repeat size during DNA replication in vitro. Expansions of up to 1000-fold were observed. Mismatches partially stabilized the repeat tracts against expansion. Expansions were only detected when the primer was complementary to the repeat tract rather than the flanking sequence. The results imply that cellular environment and whether the growing strand contains a nick or gap are important factors for the expansion process in vivo.

Step-wise DNA relaxation and decatenation by NaeI-43K.

Nae I protein was originally isolated for its restriction endonuclease properties. Nae I was later discovered to either relax or cleave supercoiled DNA, depending upon whether Nae I position 43 contains a lysine (43K) or leucine (43L) respectively. Nae I-43K DNA relaxation activity appears to be the product of coupling separate endonuclease and ligase domains within the same polypeptide. Whereas Nae I relaxes supercoiled DNA like a topoisomerase, even forming a transient covalent intermediate with the substrate DNA, Nae I shows no obvious sequence similarity to the topoisomerases. To further characterize the topoisomerase activity of Nae I, we report here that Nae I-43K changes the linking number of a single negatively supercoiled topoisomer of pBR322 by units of one and therefore is a type I topoisomerase. Positively supercoiled pBR322 was resistant to Nae I-43K. At low salt concentration Nae I-43K was processive; non-saturating amounts of enzyme relaxed a fraction of the DNA. At high salt concentration the same non-saturating amounts of Nae I-43K partially relaxed all the DNA in a step-wise fashion to give a Gaussian distribution of topoisomers, demonstrating a switch from a processive to a distributive mode of action. Nae I-43K decatenated kinetoplast DNA containing nicked circles, implying that Nae I-43K can cleave opposite a nick. The products of the reaction are decatenated nicked circles under both processive and distributive conditions. The behavior of Nae I-43K is consistent with that of a prokaryotic type I topoisomerase.

The domain organization of NaeI endonuclease: separation of binding and catalysis.

NaeI is a remarkable type II restriction endonuclease. It must bind two recognition sequences to cleave DNA, forms a covalent protein-DNA intermediate, and is only 1 aa change away from topoisomerase and recombinase activity. The latter activities apparently derive from reactivation of a cryptic DNA ligase active site. Here, we demonstrate that NaeI has two protease-resistant domains, involving approximately the N-terminal and C-terminal halves of the protein, linked by a protease-accessible region of 30 aa. The domains were purified by cloning. The C-terminal domain was shown by gel mobility-shift assay to have approximately 8-fold lower DNA-binding ability than intact NaeI. Analytical ultracentrifugation showed this domain to be a monomer in solution. The N-terminal domain, which contains the catalytic region defined by random mutagenesis, did not bind DNA and was a mixture of different-sized complexes in solution implying that it mediates self-association. DNA greatly inhibited proteolysis of the linker region. The results identify the DNA-binding domain, imply that DNA cleavage and recognition are independent and separable, and lead us to speculate about a cleft-like structure for NaeI.

Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution.

Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.

Changing a leucine to a lysine residue makes NaeI endonuclease hypersensitive to DNA intercalative drugs.

A single amino acid change transforms restriction enzyme NaeI to a topoisomerase and recombinase (NaeI-L43K) that shows no sequence similarity to these protein families. This transformation appears to result from coupled endonuclease and ligase domains. To further elucidate the relationship between NaeI-L43K and the topoisomerase protein family, we studied the effect of the topoisomerase inhibitors on NaeI-L43K activity. The intercalative drugs amsacrine, ellipticine, and daunorubicin inhibited NaeI-L43K, whereas the nonintercalating drugs camptothecin, VP-16, and oxolinic acid did not. Ethidium bromide also inhibited NaeI-L43K, implying that intercalation is responsible for its inhibition. The effects of the intercalative drugs on the DNA cleavage steps of NaeI and NaeI-L43K were compared. The drugs hardly inhibited DNA cleavage by wild type NaeI but completely inhibited DNA cleavage by NaeI-L43K. This difference in inhibition demonstrates that the L43K amino acid change sensitized NaeI to these drugs. Low concentrations of the intercalative drugs, except for ethidium bromide, enhance production of topoisomerase--DNA covalent intermediates but inhibited production of the NaeI-L43K--DNA covalent intermediate. These results imply some unique differences between DNA relaxation by NaeI-L43K and DNA topoisomerase. Concomitant with studying inhibition of the cleavage intermediate, NaeI-L43K was found to covalently bond with the 5' end of the cleaved DNA strand.

O6-methylguanine-induced replication blocks.

The ability of Klenow polymerase I, phage T7 polymerase (Sequenase), human polymerase alpha, and human polymerase beta to synthesize past (bypass) O6-methylguanine (O6-meG) lesions was studied in the presence of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to the 3' end of gel-purified oligodeoxyribonucleotide templates (45-mers), each containing a single O6-meG in place of one G in the sequence -G1G2CG3G4T-. Extension products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. A fraction of the products extended by Klenow fragment terminated either opposite or one base before O6-meG located at sites 1 and 3. Termination occurred primarily one base before O6-meG located at sites 2 and 4. The remaining fractions that bypassed the lesions represented full-length product. In control reactions, the O6-meG-containing templates were annealed with complementary 45-mers, repaired with O6-alkylguanine DNA-alkyltransferase, annealed with an excess of labeled primer, and extended by Klenow fragment. Full-length extension of > 90% was observed with each template. Primer extension past O6-meG by DNA polymerase alpha and Sequenase was partially blocked in a manner which varied with the site of O6-meG in the template while primer extension by DNA polymerase beta was completely blocked (< 2% full length extension) with O6-meG at sites 1-4. Substitution of MnCl2 for MgCl2 in the reaction mixture greatly increased the bypass of O6-meG by Klenow fragment and DNA polymerase alpha but not Sequenase or DNA polymerase beta. The increased ability of Klenow fragment to bypass O6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O6-meG at sites 1 and 2) and A (O6-meG at sites 1, 2, and 3) opposite the lesion. The results indicate that O6-meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.

DNA topoisomerase and recombinase activities in Nae I restriction endonuclease.

Nae I endonuclease must bind to two DNA sequences for cleavage. Examination of the amino acid sequence of Nae I uncovered similarity to the active site of human DNA ligase I, except for leucine 43 in Nae I instead of the lysine essential for ligase activity. Changing leucine 43 to lysine 43 (L43K) changed Nae I activity: Nae I-L43K relaxed supercoiled DNA to yield DNA topoisomers and recombined DNA to give dimeric molecules. Interruption of the reactions of Nae I and Nae I-L43K with DNA demonstrated transient protein-DNA covalent complexes. These findings imply coupled endonuclease and ligase domains and link Nae I endonuclease to the topoisomerase and recombinase protein families.

DNA cleavage by NaeI: protein purification, rate-limiting step, and accuracy.

NaeI endonuclease must bind two DNA sites for cleavage to occur. NaeI was purified to apparent homogeneity and used to determine the rate-limiting step for DNA cleavage and to measure NaeI's specificity for its cognate recognition site. Steady-state cleavage by NaeI in the presence of effector DNA (activated) gave values of 0.045 s-1 and 10 nM for kcat and KM for M13 DNA substrate, respectively, but values of 0.4 s-1 and 170 nM, respectively, for an M13 DNA fragment substrate. Single-turnover cleavage of M13 DNA demonstrated that DNA strand scission is not rate-limiting for turnover of NaeI. Transient kinetic analysis of M13 DNA cleavage by NaeI showed an initial burst of substrate cleavage that was proportional to NaeI concentration, implying that product release is rate-limiting for turnover of NaeI. The NaeI effector and substrate binding sites were found to prefer cognate over noncognate sequences by 10(3)-fold and at least 40-500-fold, respectively. kcat for noncognate recognition sequence was at least 10(6)-fold lower than that for cognate. The specificity of activated NaeI, as measured by kcat/KM, for noncognate recognition sequence was 10(8)-fold lower than that for cognate, and over 10(11)-fold lower when the decreased affinity for noncognate sequence at the effector binding site was taken into account. This specificity is approximately 10(4)-fold larger than for any other restriction enzyme measured.