Cleaving Folded RNA by Multifunctional DNAzyme Nanomachines.

Both tight and specific binding of folded biological mRNA is required for gene silencing by oligonucleotide gene therapy agents. However, this is fundamentally impossible using the conventional oligonucleotide probes according to the affinity/specificity dilemma. This study addresses this problem for cleaving folded RNA by using multicomponent agents (dubbed 'DNA nanomachine' or DNM). DNMs bind RNA by four short RNA binding arms, which ensure tight and highly selective RNA binding. Along with the improved affinity, DNM maintain the high sequence selectivity of the conventional DNAzymes. DNM enabled up to 3-fold improvement in DNAzymes catalytic efficiency (kcat/Km) by facilitating both RNA substrate binding and product release steps of the catalytic cycle. This study demonstrates that multicomponent probes organized in sophisticated structures can help to achieve the balance between affinity and selectivity in recognizing folded RNA and thus creates a foundation for applying complex DNA nanostructures derived by DNA nanotechnology in gene therapy.

RNase H-dependent DNA thresholder modulated by cancer marker concentration.

Threshold antisense oligonucleotide constructs were designed to cleave mRNA within different biomarker concentrations. The mRNA cleavage is activated by 2.6, 7.5 or 39.5 nM of biomarker depending on the construct design. The constructs can be used to differentiate cancer from normal cells by the level of oncogene expression followed by silencing of a targeted gene.

Towards the development of a DNA automaton: modular RNA-cleaving deoxyribozyme logic gates regulated by miRNAs.

Advancements in DNA computation have unlocked molecular-scale information processing possibilities, utilizing the intrinsic properties of DNA for complex logical operations with transformative applications in biomedicine. DNA computation shows promise in molecular diagnostics, enabling precise and sensitive detection of genetic mutations and disease biomarkers. Moreover, it holds potential for targeted gene regulation, facilitating personalized therapeutic interventions with enhanced efficacy and reduced side effects. Herein, we have developed six DNAzyme-based logic gates able to process YES, AND, and NOT Boolean logic. The novelty of this work lies in their additional functionalization with a common DNA scaffold for increased cooperativity in input recognition. Moreover, we explored hierarchical input binding to multi-input logic gates, which helped gate optimization. Additionally, we developed a new design of an allosteric hairpin switch used to implement NOT logic. All DNA logic gates achieved the desired true-to-false output signal when detecting a panel of miRNAs, known for their important role in malignancy regulation. This is the first example of DNAzyme-based logic gates having all input-recognizing elements integrated in a single DNA nanostructure, which provides new opportunities for building DNA automatons for diagnosis and therapy of human diseases.

Cleaving Folded RNA with DNAzyme Agents.

Cleavage of biological mRNA by DNAzymes (Dz) has been proposed as a variation of oligonucleotide gene therapy (OGT). The design of Dz-based OGT agents includes computational prediction of two RNA-binding arms with low affinity (melting temperatures (Tm ) close to the reaction temperature of 37 °C) to avoid product inhibition and maintain high specificity. However, RNA cleavage might be limited by the RNA binding step especially if the RNA is folded in secondary structures. This calls for the need for two high-affinity RNA-binding arms. In this study, we optimized 10-23 Dz-based OGT agents for cleavage of three RNA targets with different folding energies under multiple turnover conditions in 2 mM Mg2+ at 37 °C. Unexpectedly, one optimized Dz had each RNA-binding arm with a Tm ≥60 °C, without suffering from product inhibition or low selectivity. This phenomenon was explained by the folding of the RNA cleavage products into stable secondary structures. This result suggests that Dz with long (high affinity) RNA-binding arms should not be excluded from the candidate pool for OGT agents. Rather, analysis of the cleavage products' folding should be included in Dz selection algorithms. The Dz optimization workflow should include testing with folded rather than linear RNA substrates.

DNAzyme Nanomachine with Fluorogenic Substrate Delivery Function: Advancing Sensitivity in Nucleic Acid Detection.

We have developed a hook-equipped DNA nanomachine (HDNM) for the rapid detection of specific nucleic acid sequences without a preamplification step. HDNM efficiently unwinds RNA structures and improves the detection sensitivity. Compared to the hookless system, HDNM offers an 80-fold and 13-fold enhancement in DNA and RNA detection, respectively, reducing incubation time from 3 to 1 h.

Toward a Home Test for COVID-19 Diagnosis: DNA Machine for Amplification-Free SARS-CoV-2 Detection in Clinical Samples.

Nucleic acid-based detection of RNA viruses requires an annealing procedure to obtain RNA/probe or RNA/primer complexes for unwinding stable structures of folded viral RNA. In this study, we designed a protein-enzyme-free nano-construction, named four-armed DNA machine (4DNM), that requires neither an amplification stage nor a high-temperature annealing step for SARS-CoV-2 detection. It uses a binary deoxyribozyme (BiDz) sensor incorporated in a DNA nanostructure equipped with a total of four RNA-binding arms. Additional arms were found to improve the limit of detection at least 10-fold. The sensor distinguished SARS-CoV-2 from other respiratory viruses and correctly identified five positive and six negative clinical samples verified by quantitative polymerase chain reaction (RT-qPCR). The strategy reported here can be used for the detection of long natural RNA and can become a basis for a point-of-care or home diagnostic test.

The Use of Dual Energy X-Ray Bone Density Scan in Assessment of Alveolar Cleft Grafting Using Bone Marrow Stem Cells Concentrate/Platelet-Rich Fibrin Regenerative Technique.

OBJECTIVES: To determine the densitometric quality of regenerated bone at the site of bone marrow and platelet-rich membrane grafting technique at unilateral alveolar cleft region using dual energy x-ray bone density scan (DEXA). METHODS: The present prospective cohort study included 16 unilateral alveolar cleft patients who were selected randomly from the outpatient maxillofacial surgery clinic and suffered from unilateral alveolar cleft. Bone marrow aspirate and platelet-rich fibrin (PRF) (bone marrow stem cells + PRF) were used as the grafting material. Six months of follow-up have been conducted for all patients' including clinical and radiographic assessments with (DEXA scan). RESULTS: Sixteen unilateral patients with a mean age of 12.56 ±â€Š1.71 years were included in the sample and the majority of patients were females with a frequency of 56.2 percent. The current research revealed no infection or wound dehiscence. After surgery, the pain and edema scores were reasonable. Our findings showed that, after 6 months of regenerative graft, the average bone mineral density of the cleft side DEXA scan value was 1.56 ±â€Š0.32 gm/cm2, compared to 1.51 ±â€Š0.488 gm/cm2 on the normal side of the noncleft scan. There was no statistically significant difference in DEXA bone mineral content measurements between the cleft and standard sides (P = 0.461). CONCLUSIONS: The bone marrow stem cells + PRF regenerative graft technique has been successfully integrated, and the DEXA scan approach for measuring regenerated grafted bone mineral content was found to be appropriate for simple and inexpensive follow-up of alveolar cleft lip patients.

Health Care-Associated Infections in a Neurocritical Care Unit of a Developing Country.

BACKGROUND: Health care-associated infections (HAIs) in intensive care units (ICUs) specialized for neurocritical care (neurocritical care units [NCCUs]) are serious yet preventable complications that contribute significantly to morbidity and mortality worldwide. However, reliable data are scarcely available from the developing world. We aimed to analyze the incidence, epidemiology, microbial etiology, and outcomes of HAIs in an NCCU of a tertiary care teaching hospital in a high-income, developing country. METHODS: In this 3-year retrospective cohort study, all patients admitted to the NCCU at the Ibn Sina Hospital in Kuwait for ≥ 2 calendar days were included. Patient demographics, hospitalization, and details of ICU-acquired infections were evaluated. Patient-related outcomes included hospital and ICU length of stay (LOS) and in-hospital mortality. RESULTS: Among 913 patients with a total of 4921 ICU days, 79 patients had 109 episodes of HAIs. The overall incidence rate and incidence density of HAIs were 11.9/100 patients and 22.1/1000 ICU days, respectively. Multiple episodes of infection were documented in 29% of patients. The most prevalent infections were urinary tract infections (UTIs; 40/109 [37%]), bloodstream infections (30/109 [28%]), and pneumonia (16/109 [15%]). Seventy-six percent of infections were device-associated infections. A total of 158 pathogens were isolated, of which 109 were Gram-negative bacteria. Of the 40 Gram-positive bacteria, 22 were staphylococci. Seven infections were due to Clostridium difficile. There were 15 Staphylococcus aureus isolates, 47% of which were methicillin resistant. Two episodes of UTIs were due to Candida species. There were 84 Enterobacteriaceae isolates, 24% of which were extended-spectrum ß-lactamase producers. All Pseudomonas aeruginosa isolates were susceptible to aminoglycosides and carbapenems. Klebsiella species were the most common pathogen (45/158 [28%]), causing pneumonia (11/33 isolates [33%]), bloodstream infections (12/37 isolates [32%]), and UTIs (16/52 isolates [31%]). One episode of bloodstream infection was due to multidrug resistant Acinetobacter baumanii which was susceptible only to colistin. Only pneumonia was independently associated with mortality, while all HAIs that occurred were significantly associated with a prolonged ICU LOS. CONCLUSIONS: This is the first HAI surveillance study in an NCCU in Kuwait, and our results demonstrate the burden of HAIs on the neurologically injured patient, regardless of the site of infection. The high prevalence and resistant profile of HAIs in an NCCU in a developing country relative to a developed country has important implications for patient safety and emphasizes the need to strengthen collaboration between NCCU teams and infection control teams to prevent serious complications in this setting.

Surgical Management of Facial Features of Robinow Syndrome: A Case Report.

BACKGROUND: Robinow Syndrome is an extremely rare genetic disorder characterised by abnormalities in head, face and external genitalia. This disorder exists in dominant pattern with moderate symptoms and recessive pattern with more physical and skeletal abnormalities. It was first introduced by Menihard Robinow in 1969. It was related to chromosome 9q22 ROR2 gene related to bone and cartilage growth aspects. CASE PRESENTATION: A 17-year-old Egyptian male presented to National Research Centre Orodental genetics Clinic with typical features of short stature and facial dysmorphism weighted 50 Kg and measured 150 cm height complaining of facial dis figurement. There was no significant prenatal history, and family history was negative for congenital disabilities and genetic disorders. Clinical examination revealed macrocephaly and special facial features as prominent forehead, deformed ear pinna, hypertelorism, flat nasal tongue tie, deficient malar bone, bow-shaped upper and lower lips and dimpled chin. Orally the patient suffered from tonetie, gingival hypertrophy and dental malalignment. The orthopantomogram showed multiple impacted teeth. The physical examination revealed that the patient had deformed spine, short limbs with ectrodactyly, micropenis & hypospadias. Surgical management included correction of midface deficiency with zygomatic augmentation, closed rhinoplasty for the broad nose, lips muscles release and tongue tie relief. The patient is currently undergoing orthodontic treatment for his teeth. CONCLUSION: Improvement of facial features and a good psychological impact on the patient and his family.

Acute Vs. Elective Paraesophageal Hernia Repair: Endoscopic Gastric Decompression Allows Semi-Elective Surgery in a Majority of Acute Patients.

BACKGROUND: Historically, patients presenting acutely with paraesophageal hernia and requiring urgent operation demonstrated inferior outcomes compared to patients undergoing elective repair. METHODS: A prospective IRB-approved database was used to retrospectively review 570 consecutive patients undergoing paraesophageal hernia repair between 2000 and 2016. RESULTS: Thirty-eight patients presented acutely (6.7%) and 532 electively. Acute presentation was associated with increased age (74 vs. 69 years) but similar age-adjusted Charlson comorbidity scores. A history of chest pain, intrathoracic stomach ≥75%, and mesoaxial rotation were more common in acute presentations. Emergency surgery was required in three patients (8%), and 35 patients were managed in a staged approach with guided decompression prior to semi-elective surgery. Acute presentation was associated with an increased hospital stay (5 (2-13) days vs. 4 (1-27) days, p = 0.001). There was no difference in postoperative Clavien-Dindo severity scores. One patient in the elective group died, and the overall mortality was 0.2%. CONCLUSION: Our findings suggest that a majority of patients presenting with acute paraesophageal hernia can undergo a staged approach instead of urgent surgery with comparable outcomes to elective operations in high-volume centers. We suggest elective repair for patients presenting with a history of chest pain, intrathoracic stomach ≥75%, and a mesoaxial rotation.

Curcumin induces crosstalk between autophagy and apoptosis mediated by calcium release from the endoplasmic reticulum, lysosomal destabilization and mitochondrial events.

Curcumin, a major active component of turmeric (Curcuma longa, L.), has anticancer effects. In vitro studies suggest that curcumin inhibits cancer cell growth by activating apoptosis, but the mechanism underlying these effects is still unclear. Here, we investigated the mechanisms leading to apoptosis in curcumin-treated cells. Curcumin induced endoplasmic reticulum stress causing calcium release, with a destabilization of the mitochondrial compartment resulting in apoptosis. These events were also associated with lysosomal membrane permeabilization and of caspase-8 activation, mediated by cathepsins and calpains, leading to Bid cleavage. Truncated tBid disrupts mitochondrial homeostasis and enhance apoptosis. We followed the induction of autophagy, marked by the formation of autophagosomes, by staining with acridine orange in cells exposed curcumin. At this concentration, only the early events of apoptosis (initial mitochondrial destabilization with any other manifestations) were detectable. Western blotting demonstrated the conversion of LC3-I to LC3-II (light chain 3), a marker of active autophagosome formation. We also found that the production of reactive oxygen species and formation of autophagosomes following curcumin treatment was almost completely blocked by N-acetylcystein, the mitochondrial specific antioxidants MitoQ10 and SKQ1, the calcium chelators, EGTA-AM or BAPTA-AM, and the mitochondrial calcium uniporter inhibitor, ruthenium red. Curcumin-induced autophagy failed to rescue all cells and most cells underwent type II cell death following the initial autophagic processes. All together, these data imply a fail-secure mechanism regulated by autophagy in the action of curcumin, suggesting a therapeutic potential for curcumin. Offering a novel and effective strategy for the treatment of malignant cells.

Risk assessment of slow or no-reflow phenomenon in aortocoronary vein graft percutaneous intervention.

Slow or no-reflow phenomenon (SNR) complicates 10%-15% of cases of percutaneous intervention (PCI) in aortocoronary saphenous vein grafts (SVG). At present, there are no uniform, effective strategies to predict or prevent this common and potentially serious complication. The purpose of our study was to characterize variables correlated with the risk of SNR in SVG PCI in the era of stenting and glycoprotein IIb/IIIa receptor inhibitors. We identified 2,898 consecutive patients who had PCI, of whom 163 underwent PCI of at least one SVG. The clinical and angiographic characteristics of patients who developed SNR (SNR group) were compared with those who did not (no-SNR group). A total of 23 patients experienced SNR and 140 did not. Using a stepwise multivariate logistic regression analysis, four independent predictors for SNR were detected: probable thrombus (OR 6.9; 95% CI, 2.1-23.9; P = 0.001), acute coronary syndromes (OR 6.4; 95% CI, 2.0-25.3; P = 0.003), degenerated vein graft (OR 5.2; 95% CI, 1.7-16.6; P = 0.003), and ulcer (OR 3.4; 95% CI, 0.99-11.6; P = 0.04). The risk of developing SNR could be estimated according to the number of predictors found: low-grade risk (1%-10%) if < or = one variable was present, moderate risk (20%-40%) if two variables were present, and high risk (60%-90%) if three or more variables were present. We identified and quantified current risk factors for SNR and concluded that the risk of developing SNR during PCI in SVG can be predicted by simple clinical and angiographic variables obtained before PCI. This information may be useful when the risk of PCI has to be balanced against alternative strategies such as medical therapy or redo-bypass surgery or in the selection of those patients that will most benefit from the use of protection devices during PCI.

Isolated acute anterolateral papillary muscle rupture presenting as a sole manifestation of acute myocardial infarction and mimicking mitral valve vegetation.

We present here a case of an acute myocardial infarction presenting solely as rupture of the head of anterolateral papillary muscle of the mitral valve with an echocardiographic appearance of a mitral valve vegetation. A 61-year-old male patient presented to the hospital with cardiogenic shock. Transesophageal echocardiography revealed normal left ventricular global and regional systolic function with the echocardiographic appearance of a large vegetation attached to the anterior mitral valve leaflet and severe mitral regurgitation. Intraoperatively, an infracted and ruptured head of the anterolateral papillary muscle was found with no evidence of vegetations. Papillary muscle rupture is a rare complication of acute myocardial infarction, is usually associated with inferior myocardial infarction and rarely seen as the only clinical and echocardiographic finding. Transesophageal echocardiography is more sensitive than transthoracic echocardiography but misdiagnosis can still occur.

Endovascular repair of traumatic pseudoaneurysm by uncovered self-expandable stenting with or without transstent coiling of the aneurysm cavity.

Various surgical options for internal carotid or subclavian artery pseudoaneurysm repair have been reported; however, in general they have resulted in poor outcomes with high morbidity and mortality rates. Recently, these open surgical procedures have been partly replaced by percutaneous transluminal placement of endovascular devices. We evaluated the potential for using flexible self-expanding uncovered stents with or without coiling to treat extracranial internal carotid, subclavian and other peripheral artery posttraumatic pseudoaneurysm. Three patients with posttraumatic pseudoaneurysm were treated by stent deployment and coiling (two cases) of the aneurysm cavity. In one case, a 5.0 x 47 mm Wallstent (Boston Scientific) was positioned to span the neck of the 9 x 5 mm size pseudoaneurysm (left internal carotid artery) and deployed. Angiography demonstrated complete occlusion of the pseudoaneurysm without coiling. In the second patient, a 5.0 x 31 mm Wallstent (Boston Scientific) was positioned to span the neck of the 9 x 7 mm size pseudoaneurysm (right internal carotid artery) and deployed. A total of six coils (Guglielmi Detachable Coils, Boston Scientific) were deployed into the pseudoaneurysm cavity until it was completely obliterated. In the third case, an 8.0 x 80 mm SMART (Cordis) stent was advanced over the wire, positioned to span the neck of the 10 x 7 mm size pseudoaneurysm of the left subclavian artery, and deployed. Fourteen 40 x 0.5 mm Trufill (Cordis) pushable coils were deployed into the pseudoaneurysm cavity until it was completely obliterated. At long-term follow-up (6-9 months), all patients were asymptomatic without flow into the aneurysm cavity by Duplex ultrasound. We conclude that uncovered endovascular flexible self-expanding stent placement with transstent coil embolization of the pseudoaneurysm cavity is a promising new technique to treat posttraumatic pseudoaneurysm vascular disease by minimally invasive methods, while preserving the patency of the vessel and side branches.

Echocardiographic evaluation of left-ventricular diastolic function in patients with chronic pulmonary hypertension.

Different patterns of left-ventricular (LV) diastolic dysfunction were reported in patients with pulmonary hypertension (PHT). There are no data regarding the relationship between the severity of PHT and LV diastolic dysfunction. In order to determine the severity of PHT at which LV diastolic dysfunction occurs and to identify its pattern, we studied by Doppler echocardiography 120 patients with PHT (57 with severe PHT and 63 with mild or moderate PHT) and compared them with 75 normal controls. Systolic pulmonary artery pressure (SPAP) was measured by tricuspid regurgitant jet method and the usual transmitral LV diastolic indices were recorded. LV diastolic dysfunction of impaired relaxation type is most commonly seen in patients with severe PHT. No differences were observed between patients with mild and moderate PHT regarding LV diastolic function. A SPAP > or =60 mm Hg is needed to induce changes in the LV diastolic filling pattern.

Interrogation of the tricuspid annulus by Doppler tissue imaging in patients with chronic pulmonary hypertension: implications for the assessment of right-ventricular systolic and diastolic function.

BACKGROUND: Chronic pulmonary hypertension (CPHT) has a great impact on both right-ventricular (RV) systolic and diastolic properties and the assessment of those properties is not always feasible by traditional echocardiographic examination. Doppler tissue imaging (DTI) interrogation of the tricuspid annulus (TA) identifies the pattern of TA motion (TAM) and can help to assess RV function when other methods are not feasible. AIMS: To determine RV systolic and diastolic function in patients with CPHT using DTI parameters of the TA. METHODS: Eighty-seven patients with CPHT and 90 normal controls were studied. DTI parameters were measured including early diastolic, late diastolic and systolic velocities and time velocity integrals (TVI) of the TAM at both its lateral and medial aspect. RESULTS: Early diastolic and systolic velocities, the ratio of early to late diastolic velocities and TVI of TAM at both lateral and medial aspects were significantly decreased in patients with CPHT compared to controls. No significant differences were seen in late diastolic velocities and TVI in both groups. Systolic velocity of the TAM at both its lateral and medial aspects significantly correlated with RV systolic function as measured by fractional RV area change. CONCLUSIONS: DTI of the TAM can be used to assess RV systolic and diastolic properties in patients with CPHT.

Percutaneous intervention in saphenous venous grafts: in-stent restenosis lesions are safer than de novo lesions.

BACKGROUND: The histological appearance of stenosis in de novo saphenous venous grafts (DNSVG) consists of diffuse atherosclerosis that contains blood elements, necrotic debris and limited fibrocollagenous tissue. The friable nature of these lesions complicates percutaneous intervention (PCI) procedures. On the other hand, in-stent restenosis (ISR) of SVG is due primarily to atherosclerotic plaque or fibromuscular hyperplasia, with thrombus formation playing a secondary role. The purpose of this study is to compare the results of PCI in these two types of SVG lesions. METHODS: We reviewed our institutional interventional database from March 1996 through February 2000 and identified all consecutive patients who underwent PCI of at least one SVG. One hundred and ten patients were identified: 89 undergoing DNSVG intervention and 21 patients with ISR lesions. RESULTS: Acute coronary syndromes, degenerated and thrombus-containing lesions were more common in the DNSVG group. "Slow-, no-reflow" complicated 20% of the DNSVG lesions compared to none of the ISR lesions (p = 0.02). Post-procedural myocardial infarction was higher in the DNSVG group (13.5% versus 0%; p = 0.1) and correlated significantly with the occurrence of "slow-, no-reflow" (r = 0.43; p = 0.0001). Utilizing statistical modeling to adjust for baseline differences between the groups, ISR lesions were associated with a low risk of procedural complications (r = 0.22; p = 0.03). CONCLUSION: This study demonstrates that in this relatively high-risk population, PCI is safer in ISR lesions than in de novo SVG lesions.

Percutaneous and surgical interventions for in-stent restenosis: long-term outcomes and effect of diabetes mellitus.

OBJECTIVE: We examined long-term outcomes of patients with in-stent restenosis (ISR) who underwent different percutaneous interventions at the discretion of individual operators: balloon angioplasty (BA), repeat stent or rotational atherectomy (RA). We also examined long-term outcomes of patients with ISR who underwent coronary artery bypass surgery (CABG). BACKGROUND: In-stent restenosis remains a challenging problem, and its optimal management is still unknown. METHODS: Symptomatic patients (n = 510) with ISR were identified using cardiac catheterization laboratory data. Management for ISR included BA (169 patients), repeat stenting (117 patients), RA (107 patients) or CABG (117 patients). Clinical outcome events of interest included death, myocardial infarction, target vessel revascularization (TVR) and a combined end point of these major adverse cardiovascular events (MACE). Mean follow-up was 19+/-12 months (range = 6 to 61 months). RESULTS: Patients with ISR treated with repeat stent had significantly larger average post-procedure minimal lumen diameter compared with BA or RA (3.3+/-0.4 mm vs. 3.0+/-0.4 vs. 2.9+/-0.5, respectively, p < 0.05). Incidence of TVR and MACE were similar in the BA, stent and RA groups (39%, 40%, 33% for TVR and 43%, 40%, 33% for MACE, p = NS). Patients with diabetes who underwent RA had similar outcomes as patients without diabetes, while patients with diabetes who underwent BA or stent had worse outcomes than patients without diabetes. Patients who underwent CABG for ISR, mainly because of the presence of multivessel disease, had significantly better outcomes than any percutaneous treatment (8% for TVR and 23% for MACE). CONCLUSIONS: In this large cohort of patients with ISR and in the subset of patients without diabetes, long-term outcomes were similar in the BA, repeat stent and RA groups. Tissue debulking with RA yielded better results only in diabetic patients. Bypass surgery for patients with multivessel disease and ISR provided the best outcomes.

Successful treatment of coronary artery perforation in an abciximab-treated patient by microcoil embolization.

We describe a case of type 2 coronary artery perforation in a 73-year-old man undergoing coronary artery rotablation and stenting with abciximab therapy. The coronary artery perforation was successfully treated by coil embolization with Trufill pushable coils made from platinum alloy and synthetic fibers to promote maximum thrombogenicity.