A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP-PNP-DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP-PNP-DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.

Effect of Frequency of Micro-osteoperforation on Miniscrew- supported Canine Retraction: A Single-centered, Split-mouth Randomized Controlled Trial.

AIM: The present study aimed at evaluating the increase in the rate of tooth movement by increasing the number and frequency of micro-osteoperforations (MOPs). MATERIALS AND METHODS: The study was a single-center, split-mouth, randomized controlled trial. A total of 20 patients were included in the study who had fully erupted maxillary canines with class I molar canine relationship and a bimaxillary protrusion that required the removal of both maxillary and mandibular first premolars. Out of 80 samples, the experimental and controlled groups were randomly assigned. The experimental group received five MOPs in the extracted site of the first premolar before retraction, at 28th day and 56th day. The control group received no MOPs. The rate of tooth movement was measured on 28th, 56th, and 84th day on both the experimental and control sides. RESULTS: In maxillary dentition, the canine on the MOP side moved by 0.65 ± 0.21 mm, 0.74 ± 0.23 mm, and 0.87 ± 0.27 mm during 28th, 56th, and 84th day, respectively, whereas in control side the rate of tooth movement was 0.37 ± 0.09 mm, 0.43 ± 0.11 mm, and 0.47 ± 0.11 mm during 28th, 56th and 84th day, respectively, which was statistically significant (p-value = 0.000). In mandibular dentition, the canine on the MOP site has moved by 0.57 ± 0.12 mm, 0.68 ± 0.21 mm, and 0.67 ± 0.10 mm during 28th, 56th, and 84th day, respectively, whereas in control side the rate of the tooth movement was 0.34 ± 0.08 mm, 0.40 ± 0.15 mm, and 0.40 ± 0.13 mm during 28th, 56th, and 84th day, respectively, which was statistically significant. CONCLUSION: Micro-osteoperforations effectively increased the rate of tooth movement. Overall, MOPs increased the rate of canine retraction by 2-fold when compared with the control group. CLINICAL SIGNIFICANCE: Micro-osteoperforation is a proven methodology to increase the rate of tooth movement and decrease the treatment time. However, it is important to repeat the procedure during every activation to increase its effectiveness.

Non-radiation and non-drug-induced maxillary osteomyelitis: Study of underlying risk factors, presentation, management and treatment outcomes.

Background: Osteomyelitis of the jawbone is mostly secondary to radiation exposure or bone remodelling drugs, with the mandible being commonly involved. Maxillary osteomyelitis risk is low owing to its high vascularity. This study was undertaken to evaluate risk factors, presentation, management and outcomes of maxillary osteomyelitis caused due to reasons other than irradiation and bone remodelling drugs. Methods: Patient records diagnosed with maxillary osteomyelitis were evaluated for demographic details, risk factors, clinical presentation, radiological features, treatment performed and outcomes. Results: In 38 patients with non-irradiated and non-drug-induced osteomyelitis, 13 involved the maxilla, seven were localized to the posterior maxilla and 10 showed paranasal sinus involvement. Dissemination to the cavernous sinus and cerebral spread was seen in one. Clinical findings included oroantral communication, pain and draining sinus. Imaging showed diffuse bone destruction areas with or without evidence of bony sequestrum. The most common systemic risk factor was diabetes mellitus. Maxillary osteomyelitis was associated with tooth extraction in eight cases. Surgical management included debridement, sequestrectomy, functional endoscopic sinus surgery, maxillectomy and reconstruction of soft tissue defect with local and regional flaps. Complete recovery was seen in 11 patients. Mortality was seen in two patients with mucormycosis having disseminated infection. Conclusion: Compared with previous literature, a relatively higher ratio of maxillary involvement was reported. Diabetes mellitus was the most common risk factor, followed by osteopetrosis and tooth extraction. Osteomyelitis secondary to mucormycosis in immunocompetent patients was relatively localized and gave favourable response to management compared with patients with diabetes mellitus.

Nucleotide-Induced Conformational Changes in Escherichia coli DnaA Protein Are Required for Bacterial ORC to Pre-RC Conversion at the Chromosomal Origin.

DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC) to a replication efficient pre-replication complex (pre-RC) at the E. coli chromosomal origin of replication (oriC).

Current Advances in Graft-versus-host Disease After Intestinal Transplantation.

Graft-versus-host disease (GvHD) remains a potentially fatal complication following intestinal transplant (ITx). Over the past decade, advances in the understanding of the pathophysiology of this complex immunological phenomenon have led to the reassessment of the host systemic immune response and have created a gateway for novel preventive and therapeutic strategies. Although sufficient evidence dictates the use of corticosteroids as a first-line option, the treatment for refractory disease remains contentious and lacks a standardized therapeutic approach. Timely diagnosis remains crucial, and the advent of chimerism detection and immunological biomarkers have transformed the identification, prognostication, and potential for survival after GvHD in ITx. The objectives of the following review aim to discuss the clinical and diagnostic features, pathophysiology, advances in immune biomarkers, as well as therapeutic opportunities in the prevention and treatment of GvHD in ITx.

Suppressor T helper type 17 cell responses in intestinal transplant recipients with allograft rejection.

BACKGROUND: Intestinal transplant (ITx) rejection is associated with memory T helper type 17 cell (Th17) infiltration of grafted tissues. Modulation of Th17 effector cell response is facilitated by T regulatory (Treg) cells, but a phenotypic characterization of this process is lacking in the context of allograft rejection. METHODS: Flow cytometry was performed to examine the expression of surface receptors, cytokines, and transcription factors in Th17 and Treg cells in ITx control (n = 34) and rejection patients (n = 23). To elucidate key pathways guiding the rejection biology, we utilized RNA sequencing (RNAseq) and assessed epigenetic stability through pyrosequencing of the Treg-specific demethylated region (TSDR). RESULTS: We found that intestinal allograft rejection is characterized by Treg cellular infiltrates, which are polarized toward Th17-type chemokine receptor, ROR-γt transcription factor expression, and cytokine production. These Treg cell subsets have maintained epigenetic stability, as defined by FoxP3-TSDR methylation status, but displayed upregulation of functional Treg and purinergic signaling genes by RNAseq analysis such as CD39, in keeping with suppressor Th17 properties. CONCLUSION: We show that ITx rejection is associated with increased polarized cells that express a Th17-like phenotype concurrent with regulatory purinergic markers.

Circulating, cell-free methylated DNA indicates cellular sources of allograft injury after liver transplant.

Post-transplant complications reduce allograft and recipient survival. Current approaches for detecting allograft injury non-invasively are limited and do not differentiate between cellular mechanisms. Here, we monitor cellular damages after liver transplants from cell-free DNA (cfDNA) fragments released from dying cells into the circulation. We analyzed 130 blood samples collected from 44 patients at different time points after transplant. Sequence-based methylation of cfDNA fragments were mapped to patterns established to identify cell types in different organs. For liver cell types DNA methylation patterns and multi-omic data integration show distinct enrichment in open chromatin and regulatory regions functionally important for the respective cell types. We find that multi-tissue cellular damages post-transplant recover in patients without allograft injury during the first post-operative week. However, sustained elevation of hepatocyte and biliary epithelial cfDNA beyond the first week indicates early-onset allograft injury. Further, cfDNA composition differentiates amongst causes of allograft injury indicating the potential for non-invasive monitoring and timely intervention.

Crosstalk between DnaA protein, the initiator of Escherichia coli chromosomal replication, and acidic phospholipids present in bacterial membranes.

Anionic (i.e., acidic) phospholipids such as phosphotidylglycerol (PG) and cardiolipin (CL), participate in several cellular functions. Here we review intriguing in vitro and in vivo evidence that suggest emergent roles for acidic phospholipids in regulating DnaA protein-mediated initiation of Escherichia coli chromosomal replication. In vitro acidic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA to replicatively proficient ATP-DnaA, yet both PG and CL also can inhibit the DNA-binding activity of DnaA protein. We discuss how cellular acidic phospholipids may positively and negatively influence the initiation activity of DnaA protein to help assure chromosomal replication occurs once, but only once, per cell-cycle. Fluorescence microscopy has revealed that PG and CL exist in domains located at the cell poles and mid-cell, and several studies link membrane curvature with sub-cellular localization of various integral and peripheral membrane proteins. E. coli DnaA itself is found at the cell membrane and forms helical structures along the longitudinal axis of the cell. We propose that there is cross-talk between acidic phospholipids in the bacterial membrane and DnaA protein as a means to help control the spatial and temporal regulation of chromosomal replication in bacteria.

Comparative Evaluation of Effect of Micro-Osteoperforation and Mechanical Vibration on Rate of Orthodontic Tooth Movement in Young Adults With Bimaxillary Protrusion.

AIM: To evaluate and compare the rate of orthodontic tooth movement and root resorption by micro-osteoperforation (MOP) and mechanical vibration in young adults with bimaxillary protrusion. METHOD: Twenty patients having class I bimaxillary protrusion who required all first premolar extraction were allocated into two groups MOP (Group A) and mechanical vibration (Group B), with a 1:1 allocation ratio. After leveling alignment MOP was performed on either side of the arch, and vibration was applied on the contralateral side 20 mins per day. Canines were retracted with nickel-titanium coil springs, and Alginate impressions were taken every four weeks till 4 months. RESULT: The mean rate of retraction of canines of Group A was more than Group B. There was a statistically significant difference between Group A and Group B. (p=0.0120) Conclusion: The mean rate of retraction of canines treated by MOP was 1.15 mm per 4 weeks, and by mechanical vibration, 0.8mm per 4 weeks.

Type 1 Innate Lymphoid Cells Are Proinflammatory Effector Cells in Ischemia-Reperfusion Injury of Steatotic Livers.

Innate lymphoid cells (ILCs), the most recently described family of lymphoid cells, play fundamental roles in tissue homeostasis through the production of key cytokine. Group 1 ILCs, comprised of conventional natural killer cells (cNKs) and type 1 ILCs (ILC1s), have been implicated in regulating immune-mediated inflammatory diseases. However, the role of ILC1s in nonalcoholic fatty liver disease (NAFLD) and ischemia-reperfusion injury (IRI) is unclear. Here, we investigated the role of ILC1 and cNK cells in a high-fat diet (HFD) murine model of partial warm IRI. We demonstrated that hepatic steatosis results in more severe IRI compared to non-steatotic livers. We further elicited that HFD-IRI mice show a significant increase in the ILC1 population, whereas the cNK population was unchanged. Since ILC1 and cNK are major sources of IFN-γ and TNF-α, we measured the level of ex vivo cytokine expression in normal diet (ND)-IRI and HFD-IRI conditions. We found that ILC1s in HFD-IRI mice produce significantly more IFN-γ and TNF-α when compared to ND-IRI. To further assess whether ILC1s are key proinflammatory effector cells in hepatic IRI of fatty livers, we studied both Rag1-/- mice, which possess cNK cells, and a substantial population of ILC1s versus the newly generated Rag1-/-Tbx21-/- double knockout (Rag1-Tbet DKO) mice, which lack type 1 ILCs, under HFD IRI conditions. Importantly, HFD Rag1-Tbet DKO mice showed significant protection from hepatic injury upon IRI when compared to Rag1-/- mice, suggesting that T-bet-expressing ILC1s play a role, at least in part, as proinflammatory effector cells in hepatic IRI under steatotic conditions.

Oral N-acetylcysteine decreases IFN-γ production and ameliorates ischemia-reperfusion injury in steatotic livers.

Type 1 Natural Killer T-cells (NKT1 cells) play a critical role in mediating hepatic ischemia-reperfusion injury (IRI). Although hepatic steatosis is a major risk factor for preservation type injury, how NKT cells impact this is understudied. Given NKT1 cell activation by phospholipid ligands recognized presented by CD1d, we hypothesized that NKT1 cells are key modulators of hepatic IRI because of the increased frequency of activating ligands in the setting of hepatic steatosis. We first demonstrate that IRI is exacerbated by a high-fat diet (HFD) in experimental murine models of warm partial ischemia. This is evident in the evaluation of ALT levels and Phasor-Fluorescence Lifetime (Phasor-FLIM) Imaging for glycolytic stress. Polychromatic flow cytometry identified pronounced increases in CD45+CD3+NK1.1+NKT1 cells in HFD fed mice when compared to mice fed a normal diet (ND). This observation is further extended to IRI, measuring ex vivo cytokine expression in the HFD and ND. Much higher interferon-gamma (IFN-γ) expression is noted in the HFD mice after IRI. We further tested our hypothesis by performing a lipidomic analysis of hepatic tissue and compared this to Phasor-FLIM imaging using "long lifetime species", a byproduct of lipid oxidation. There are higher levels of triacylglycerols and phospholipids in HFD mice. Since N-acetylcysteine (NAC) is able to limit hepatic steatosis, we tested how oral NAC supplementation in HFD mice impacted IRI. Interestingly, oral NAC supplementation in HFD mice results in improved hepatic enhancement using contrast-enhanced magnetic resonance imaging (MRI) compared to HFD control mice and normalization of glycolysis demonstrated by Phasor-FLIM imaging. This correlated with improved biochemical serum levels and a decrease in IFN-γ expression at a tissue level and from CD45+CD3+CD1d+ cells. Lipidomic evaluation of tissue in the HFD+NAC mice demonstrated a drastic decrease in triacylglycerol, suggesting downregulation of the PPAR-γ pathway.

Membrane Stress Caused by Unprocessed Outer Membrane Lipoprotein Intermediate Pro-Lpp Affects DnaA and Fis-Dependent Growth.

In Escherichia coli, repression of phosphatidylglycerol synthase A gene (pgsA) lowers the levels of membrane acidic phospholipids, particularly phosphatidylglycerol (PG), causing growth-arrested phenotype. The interrupted synthesis of PG is known to be associated with concomitant reduction of chromosomal content and cell mass, in addition to accumulation of unprocessed outer membrane lipoprotein intermediate, pro-Lpp, at the inner membrane. However, whether a linkage exists between the two altered-membrane outcomes remains unknown. Previously, it has been shown that pgsA + cells overexpressing mutant Lpp(C21G) protein have growth defects similar to those caused by the unprocessed pro-Lpp intermediate in cells lacking PG. Here, we found that the ectopic expression of DnaA(L366K) or deletion of fis (encoding Factor for Inversion Stimulation) permits growth of cells that otherwise would be arrested for growth due to accumulated Lpp(C21G). The DnaA(L366K)-mediated restoration of growth occurs by reduced expression of Lpp(C21G) via a σ E -dependent small-regulatory RNA (sRNA), MicL-S. In contrast, restoration of growth via fis deletion is only partially dependent on the MicL-S pathway; deletion of fis also rescues Lpp(C21G) growth arrest in cells lacking physiological levels of PG and cardiolipin (CL), independently of MicL-S. Our results suggest a close link between the physiological state of the bacterial cell membrane and DnaA- and Fis-dependent growth.

Kimura's Disease without Peripheral Eosinophilia: An Unusual and Challenging Case Simulating Venous Malformation on Imaging Studies-Case Report and Review of literature.

Kimura's Disease (KD) is a rare chronic inflammatory disorder presenting as multiple painless solitary subcutaneous nodules, predominantly in the head and neck region and frequently associated with regional lymphadenopathy and/or salivary gland involvement. Because of painless nature and indolent course, there is usually a delay in the patient's presentation. KD may radiologically mimic other chronic inflammatory conditions like tuberculosis, vascular malformations and neoplasms. Clinical correlation and histological evaluation along with elevated peripheral eosinophil and serum IgE level are considered important for confirmatory diagnosis. We report a case of painless swelling over right submandibular region extending to the right superficial parotid. The haematological reports were within normal limits. Ultrasound (USG), Magnetic Resonance Imaging (MRI) and Magnetic Resonance Angiogram (MRA) favoured a diagnosis of venous malformation. However, histopathological examination of excised lesion confirmed a diagnosis of KD. This case proves the possibility of the KD even in the absence of peripheral eosinophilia and/ or elevated serum IgE level, and may mimic venous malformation on imaging studies. Therefore, KD must find a place in the differentials of solitary painless neck swelling even in the absence of peripheral eosinophilia and/or elevated IgE level.

Role of PET-CT Scan in Gynaeconcology.

THE PURPOSE OF THIS STUDY: This study was undertaken to evaluate the role of positron emission tomography-CT (PET-CT) in diagnosis and management of gynecological malignancies in primary and recurrent settings and also to investigate its role in inappropriately treated patients, for pretreatment evaluation (staging) to help in proper therapeutic management. METHOD: This is a retrospective study of 56 patients of gynecological malignancy registered in Gujarat Cancer Research Institute from June 2011 to December 2013. RESULTS: Out of 56 cases where PET was done, the results were as follows: carcinoma cervix-23, carcinoma ovary-20, carcinoma endometrium-9, carcinoma vulva-1, carcinoma vagina-2, and GTN-1. PET scan was negative in 37 % of patients where CT scan was suspicious, which changed the therapeutic modality and prevented further unnecessary interventions. In cases where clinical suspicion of recurrence was high based on rising tumor marker and CT scan was negative, subsequent PET-CT was able to pick up malignancy in 75 % cases. Eleven patients (25 %) with suspected recurrence with inconclusive CT scan and negative PET-CT scan were kept on follow-up, thus reducing further morbidity and cost. CONCLUSION: Addition of PET-CT, a noninvasive method to the oncologist's imaging armamentarium may ultimately improve both outcomes and costs by altering management strategies in primary and recurrent settings. The potential use of PET-CT appears promising in several decision-making steps in the management of patients with gynecological malignancy. It defines the extent of metastatic disease which enables the clinician to decide regarding salvageable surgical intervention or palliative measures.