High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics.

To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next-generation sequencing data. By using this approach, we sequenced longitudinally collected acute myeloid leukemia (AML) tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify cells harboring pathogenic mutations during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of AML heterogeneity, leading to improved stratification and therapy selection for the disease.

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity.

The RNA-binding heme protein DiGeorge critical region 8 (DGCR8) and its ribonuclease partner Drosha cleave primary transcripts of microRNA (pri-miRNA) as part of the canonical microRNA (miRNA) processing pathway. Previous studies show that bis-cysteine thiolate-coordinated Fe(III) DGCR8 supports pri-miRNA processing activity, while Fe(II) DGCR8 does not. In this study, we further characterized Fe(II) DGCR8 and tested whether CO or NO might bind and restore pri-miRNA processing activity to the reduced protein. Fe(II) DGCR8 RNA-binding heme domain (Rhed) undergoes a pH-dependent transition from 6-coordinate to 5-coordinate, due to protonation and loss of a lysine ligand; the ligand bound throughout the pH change is a histidine. Fe(II) Rhed binds CO and NO from 6- and 5-coordinate states, forming common CO and NO adducts at all pHs. Fe(II)-CO Rhed is 6-coordinate, low-spin, and pH insensitive with the histidine ligand retained, suggesting that the protonatable lysine ligand has been replaced by CO. Fe(II)-NO Rhed is 5-coordinate and pH insensitive. Fe(II)-NO also forms slowly upon reaction of Fe(III) Rhed with excess NO via a stepwise process. Heme reduction by NO is rate-limiting, and the rate would be negligible at physiological NO concentrations. Importantly, in vitro pri-miRNA processing assays show that both CO- and NO-bound DGCR8 species are inactive. Fe(II), Fe(II)-CO, and Fe(II)-NO Rhed do not bear either of the cysteine ligands found in the Fe(III) state. These data support a model in which the bis-cysteine thiolate ligand environment of Fe(III) DGCR8 is necessary for establishing proper pri-miRNA binding and enabling processing activity.

Preprocedural statin therapy, inflammation, and myocardial injury in low-risk stable coronary artery disease patients submitted to coronary stent implantation.

OBJECTIVE: Evaluate if statin therapy prior to elective coronary stent implantation (CSI) reduces the plasma levels of markers of inflammation and of myocardial necrosis in low-risk stable coronary artery disease patients (CAD). BACKGROUND: The elevation of markers of inflammation and of myocardial necrosis after percutaneous coronary intervention may interfere with clinical outcome. Among acute coronary syndrome patients, statins improve clinical outcomes when used before CSI-mostly due to reduction of CSI-related myocardial infarction. However, little is known concerning preprocedural statin therapy on the reduction of these markers in stable patients at low-risk. METHODS: In this prospective, observational study, 100 patients (n = 50 on statin therapy vs. n = 50 not on statin) with stable coronary artery disease underwent elective CSI. Inflammatory (C-reactive protein [CRP], interleukin [IL]-6, tumor necrosis factor-α and matrix metalloproteinase-9) and myocardial necrosis markers (troponin I and CK-MB) were determined before and 24 hr after CSI. RESULTS: All patients presented a significant increase of CRP and IL-6 after CSI. However, this increase was attenuated in patients on statin therapy prior to CSI than those without statin therapy: 75% vs. 150% (P < 0.001) and 192% vs. 300% (P < 0.01). The other pro-inflammatory markers were similar for both sets of patients. Troponin I and CK-MB did not change after CSI regardless of previous statin therapy or not. CONCLUSIONS: Pretreatment with statin attenuates procedural inflammation, denoted by markedly lower increases of CRP and IL-6 levels, in elective CSI within low-risk stable CAD patients. Periprocedural myocardial injury was irrelevant and was not affected by preprocedural statin therapy in this population.

Anatomical variations and embryological basis of arch of aorta and aortic valve.

Variations in the arch of the aorta and aortic valves among fetal, cadaveric, and post-mortem specimens present a spectrum of anatomical configurations, posing challenges in establishing a standard norm. While some variations hold surgical significance, many bear little functional consequence but provide insights into embryological origins. The aortic arch exhibits diverse branching patterns, including common trunks and different orders, relevant for endovascular surgeries. Meanwhile, malformations in the aortic valve, affecting the aorta, may lead to ischemia and cerebral infarction, warranting understanding of coexisting arch and valve anomalies to predict complications like aortic dissection. Studies in the Indian population mirror global variations, underscoring the need to explore embryological, clinical, and surgical implications for safer vascular surgeries involving the aortic arch and valves. The study's objectives included examining branching patterns, diameters, and distances between arch branches and exploring aortic valve variations. Employing a cross-sectional design, the study was conducted across Anatomy, Forensic Medicine, and Obstetrics and Gynecology departments. A sample of 100, comprising cadavers, fetuses, and postmortem specimens, were gathered. Specimens ranged from 14 weeks of intrauterine life to 85 years, with intact thoracic cages as inclusion criteria. Methodology involved dissection, specimen fixation, and macroscopic examination for variations and morphological parameters. Results showed aortic diameter increase with age, with significant gender differences. A statistically significant association between arch variations and anomalous valves was observed, suggesting mutual predictability. Individuals with valve anomalies should undergo comprehensive cardiology evaluation to avert complications like aortic dissection during endovascular surgeries. While atheromatous plaques were prevalent in younger groups, their frequency rose with age, necessitating vigilant vascular monitoring. Careful handling during surgeries is paramount, given potential adverse outcomes resulting from variations. Overall, the study underscores the importance of comprehensive anatomical understanding in clinical contexts, guiding effective management strategies and ensuring patient safety in vascular surgeries.

Comparative Evaluation of Microleakage of Zinc Phosphate Cement, Resin-Modified Glass Ionomer, and Two Dual-Cure Resin Cements: In Vitro Study.

Aims: The aim of this study is to evaluate and compare the sealing ability of a new resin cement which was undergoing trial with other three luting cements. Settings and Design: In vitro study. Subjects and Methods: Forty extracted intact human premolars were selected and standardized class 5 preparations for inlays were done at cementoenamel junction of buccal surfaces and direct composite inlay was fabricated. The samples were randomly grouped into 4: Group I luted with Rely X ARC resin cement, Group II with DCRC-10, a new indigenously developed resin cement, Group III with FujiCEM, and Group IV with zinc phosphate. After 24 h storage, samples were thermocycled and immersed in basic fuchsine dye. The longitudinal sections were assessed under a stereomicroscope for microleakage using graded criteria. Statistical Analysis Used: To analyze leakage scores among four groups and between enamel and dentin/cementum margins, Kruskal-Wallis nonparametric test and the Mann-Whitney test at P < 0.05 were used. Results: The dye penetration at cementum/dentin margins showed a statistically significant difference among four groups (P < 0.001). Adhesive luting cements produced lesser leakage scores at both margins than those cemented with zinc phosphate. Conclusions: The indigenously developed resin cement (DCRC-10, Group II) is comparable to that of Group I (RelyX ARC) in terms of its luting efficiency.

Left cervical aortic arch associated with multiple vascular anomalies.

Cervical aortic arch is a rare anomaly occasionally associated with other cardiovascular abnormalities. We present a case of tortuous left cervical aortic arch associated with hypoplastic transverse arch, coarctation of the aorta, and right brachiocephalic arteries arising below the coarctation and stenotic origin of the left subclavian artery. These multiple anatomic anomalies, which are associated in our case, have not been described in a single patient previously.

Effects of climate change and anthropogenic pressures in the water quality of a coastal lagoon (Ria Formosa, Portugal).

Understanding how climatic and anthropogenic drivers will influence coastal lagoons is fundamental to guarantee their preservation and sustainability. The Ria Formosa (coastal lagoon, South coast of Portugal) is a very important ecosystem that supports diverse economic activities in the region. The 3D coupled hydrodynamic-biogeochemical model SCHISM was validated and used to assess the influence of climate change and anthropogenic pressures on the water quality of the Ria Formosa. Five scenarios were simulated: reference scenario (S0), mean sea level rise (SLR) of 0.5 m (S1), increase of the air temperature of 1.68 °C (S2), increase of the outflow from the wastewater treatment plants (WWTP) by 50% (S3) and a combined scenario (S4). Results suggest that SLR of 0.5 m promotes an increase of 0.5-3 in the salinity near the area of influence of the WWTP. SLR decreases the inorganic nutrient concentrations in these areas by about 40-60%, due to an increase of the dilution. In contrast, the increase of the outflow from the WWTP by 50% increases the nutrients concentrations by about 20-40%. The increase of the air temperature alone by 1.68 °C increases the water temperature by 0-1 °C. The combined scenario suggests antagonist effects in the nutrient concentrations. Overall, the trophic index (TRIX) of the lagoon calculated for the scenarios exhibits only minor differences relative to the reference scenario, except in some areas near the WWTP discharges. In these areas, TRIX tends to increase with the increase of the outflow from the WWTP in scenario S3. These results provide further insight into the response of coastal lagoons, and the Ria Formosa in particular, to future changes and contribute to support their management.

Mandibular Canine Calcification Stages as an Indicator of Skeletal Maturity.

AIMS: The aim of this study was to verify whether there is significant relationship between stages of mandibular canine calcification and skeletal maturation and to determine whether the same can be used as a reliable diagnostic aid to assess skeletal maturity. MATERIALS AND METHODS: The sample consisted of 50 males and 50 females aged between 8 and 15 years. Two radiographs namely orthopantomograph and handwrist radiograph were taken for this study. Skeletal age was ascertained from handwrist radiographs according to the method introduced by Fishman L.S. The developmental stages of mandibular canine were assessed according to Demirjian's stages of dental calcification. RESULTS: The relationship between canine stage calcification and skeletal maturity index was statistically significant in all stages. INTERPRETATION AND CONCLUSION: Mandibular canine calcification stages can be recommended as a supplemental diagnostic aid. Similar studies may be conducted in different population to rule out any possible racial and regional influences on growth characteristics.

Evaluation of Long-term Stability of Bilateral Sagittal Split Osteotomy in Individuals with Different Growth Patterns.

OBJECTIVE: Bilateral sagittal split osteotomy (BSSO) is a routinely used surgical step for the correction of a class III with mandibular prognathism. Many factors influence the stability of the surgical correction achieved. This study was designed to access the role of growth pattern in the surgical stability after a BSSO correction. MATERIALS AND METHODS: A total of 18 individuals (6 vertical growers, 6 horizontal growers, and 6 normal growing individuals) were considered for the study. Five parameters, horizontal plane (HP)-pogonion (POG) angle, HP-occlusal plane angle, POG height, POG depth, and Point B depth, were measured and compared postsurgically and in the follow-up phase. RESULT: Vertical growing individuals showed greater tendency for relapse and clockwise rotation of mandible postsurgically. No major difference was observed in the normal and horizontal growing individuals. When planning BSSO on vertical growing patient, utmost care should be taken to prevent posttreatment relapse to ensure better surgical stability.

Comparison of Marginal Accuracy of Interim Restoration Fabricated from Self-cure Composite and Autopolymerizing Acrylic Resin: An In Vitro Study.

BACKGROUND AND OBJECTIVES: One of the important steps toward achieving a successful fixed partial denture treatment is the fabrication of interim restorations with good marginal adaptation. Poor marginal adaptation of the interim restorations will allow ingress of fluids and microorganisms between the restorations and may lead to caries and pulpitis. The objective of this study was to evaluate and compare the marginal accuracy of interim restorations made from two polymethyl methacrylate (PMMA) and two bis-acryl materials. MATERIALS AND METHODS: An in vitro method was used to replicate a clinical procedure in which the interim crown was made using a direct technique. A maxillary first molar typodont tooth was prepared to receive a full cast crown restoration having 1 mm chamfer and a taper of 5°, and then the prepared artificial tooth was cast into a cobalt chromium metal die. Wax patterns were prepared on the metal die using type 2 inlay wax for full metal crowns. The impression of the die along with the wax pattern was made with a putty-type polyvinyl siloxane, and this impression was used as a mold for making interim restorations. Two PMMA and two bis-acryl provisional crown materials were distributed as follows: Group A: Unifast Trad (GC Dental), Group B: DPI self-cure tooth molding material, Group C: Structur 2 QM (VOCO), and Group D: Cool Temp Natural (Coltene/Whaledent). A total of 60 interim restorations from all the four materials were fabricated (15 interim restorations from each material). The marginal discrepancy of the interim restoration was determined by measuring the space between the margin of the interim restoration and the finish line of the metal die at four vertical reference lines previously marked at the midpoint of the metal die finish line at the buccal, palatal, mesial, and distal surfaces of the die. The marginal gaps were visualized at ×45 magnification using a stereomicroscope and interpreted using ProgRes image capture software. RESULTS: The mean marginal discrepancy calculated in PMMA materials was 269 µm for DPI and 205 µm for Unifast Trad, and the mean marginal gap for the bis-acryl material was 171 µm for Cool Temp and 140 µm for Structur 2 QM. INTERPRETATION AND CONCLUSION: Comparison of marginal accuracy showed that bis-acryl provisional crown material showed better marginal accuracy than PMMA provisional crown material. Among the materials tested, Structur 2 QM showed the best marginal accuracy, and the least marginal accuracy was shown by DPI.

High throughput single-cell detection of multiplex CRISPR-edited gene modifications.

CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.

Comparative Assessment of Force Decay of the Elastomeric Chain With the Use of Various Mouth Rinses in Simulated Oral Environment: An In Vitro Study.

AIM: This study is aimed to assess the effect of different mouth rinses and its active components on the force decay of elastomeric chains. Listerine, Colgate Phos-Flur, Clohex Plus mouth rinses, 26.9% alcohol, 0.04% sodium fluoride (NaF), and 0.2% chlorhexidine were used for this study to determine its effects on force decay of elastomeric chains. MATERIALS AND METHODS: Seven custom-made jigs were constructed on which 120 short module elastomeric chains were stretched to predetermined lengths between the pins. Using calibrated digital force tester, measurements of force exerted by the elastomeric chains while stretched on the framework were recorded at the time of attachment to the frame and after 24 hours, 7 days, 14 days, 21 days, and 28 days. The custom-made jigs and the elastomeric chains were allowed for complete submersion in artificial saliva throughout the test period, as well as in respective control solution and mouth rinses for 60 seconds, twice daily. RESULTS: All test groups showed significantly more force decay than the control group. Statistically significant differences were observed when comparing force decay among the test groups except between a few. INTERPRETATION AND CONCLUSION: Mouth rinses cause an increase in force decay of elastomeric chain over the time. Listerine and 26.9% alcohol solution caused maximum force decay by the end of 28 days. Least force degradation of elastomeric chain was seen with the use of 0.2% chlorhexidine.

Comparative Evaluation of Antibiofilm Efficacy of Chitosan Nanoparticle- and Zinc Oxide Nanoparticle-Incorporated Calcium Hydroxide-Based Sealer: An In vitro Study.

AIM: This study evaluated the antibiofilm efficacy of calcium hydroxide-based sealer incorporated with chitosan nanoparticles (CS-NPs) and zinc oxide nanoparticles (ZnO-NPs) against two strains of Enterococcus faecalis (ATCC 29212, OG1RF). MATERIALS AND METHODS: The materials tested were Apexit Plus sealer in the commercial unmodified form and two modified forms with CS-NP and ZNO-NP, respectively. Crystal violet assay and confocal laser scanning microscopy (CLSM) study were used to assess the bacterial viability of biofilms grown in wells of microtiter plate and glass slides, respectively. Two E. faecalis strains (ATCC 29212, OG1RF) were used for the study. RESULTS: The crystal violet assay done on E. faecalis strain ATCC 29212 showed a significant decrease in the optical density (OD) value with ZNO-NP-incorporated calcium hydroxide sealer when compared with CS-NP. In the case of E. faecalis strain OG1RF, only ZNO-NP-incorporated calcium hydroxide-based sealer showed reduction in the OD value. In CLSM study done on E. faecalis strain ATCC 29212, only ZNO-NP-incorporated calcium hydroxide-based sealer showed reduction in the thickness of biofilm. No groups of OG1RF strain showed reduction in the thickness of biofilm. CONCLUSION: The incorporation of nanoparticles (ZnO and CS) into calcium hydroxide-based sealers significantly enhances the antibiofilm efficiency against E. faecalis strain ATCC 29212 but has questionable effectiveness against E. faecalis strain OG1RF. The present study demonstrates that ZNO-NP shows better antibiofilm efficacy than CS-NP against both strains of E. faecalis.

Fasting Insulin is Better Partitioned according to Family History of Type 2 Diabetes Mellitus than Post Glucose Load Insulin of Oral Glucose Tolerance Test in Young Adults.

INTRODUCTION: Hyperinsulinemia is contributed by insulin resistance, hepatic insulin uptake, insulin secretion and rate of insulin degradation. Family history of type 2 diabetes mellitus has been reported to cause hyperinsulinemia. AIM: Correlation of fasting insulin with post glucose load Oral Glucose Tolerance Test (OGTT) insulin in young adults and their partitioning according to family history of type 2 diabetes. MATERIALS AND METHODS: In this observational cross-sectional study, clinical evaluation and biochemical assays of insulin and diabetes related parameters, and secondary clinical influences on type 2 diabetes in volunteers were done for inclusion as participants (n=90) or their exclusion. Cut off levels of quantitative biochemical variables were fixed such that they included the effects of insulin resistance, but excluded other secondary clinical influences. Distribution was analysed by Shapiro-Wilk test; equality of variances by Levene's test; Log10 transformations for conversion of groups to Gaussian distribution and for equality of variances in the groups compared. When the groups compared had Gaussian distribution and there was equality of variance, parametric methods were used. Otherwise, non parametric methods were used. RESULTS: Fasting insulin was correlating significantly with 30, 60 and 120 minute OGTT insulin showing that hyperinsulinemia in the fasting state was related to hyperinsulinemia in the post glucose load states. When fasting and post glucose load OGTT insulin were partitioned into those without and with family history of type 2 diabetes, maximum difference was seen in fasting insulin (p<0.001), followed by 120 (p=0.001) and 60 (p= 0.002) minute OGTT insulin. The 30 minute insulin could not be partitioned (p=0.574). CONCLUSION: Fasting, 60 and 120 minute OGTT insulin can be partitioned according to family history of type 2 diabetes, demonstrating stratification and heterogeneity in the insulin sample. Of these, fasting insulin was better partitioned and could be used for baseline reference interval calculations.

Reduction of urinary thiols in nephrotic syndrome--a possible effect of free iron.

BACKGROUND: Albumin is a potent antioxidant as it chelates transitional metals and contains antioxidants like thiol and bilirubin. In neprotic syndrome, the defining parameter is proteinuria with hypoalbuminemia. Therefore albuminuria in nephrotic syndrome may increase toxic transitional metal ions and also can cause loss of albumin associated antioxidants causing oxidative stress to the individual. METHODS: We investigated this possibility and estimated some markers of oxidative stress in 20 nephrotic syndrome patients and healthy controls along with urinary thiols, urinary bilirubin and plasma free iron in both cases and in the controls. RESULT: We found oxidative stress in 20 nephrotic syndrome patients and the markers of oxidative stress correlated significantly with proteinuria, but the urine of nephrotic syndrome patients (28.33+/-4.2 micromol/g creatinine)contained significantly less thiols compared to the healthy controls (88.45+/-10.6 micromol/g creatinine) and no biliribin. The patients plasma also showed free iron (0.7+/-0.05 micromol/l), a parameter undetectable in the healthy controls. CONCLUSION: We suggest that oxidative stress and presence of free iron in the patients were responsible for less thioluria and no bilirubinuria. A detailed study of oxidative biology in a large cohort of nephrotic syndrome patients is necessary to confirm the presence of free iron as appropriate chelation of free iron may benefit the long-term prognosis of the disease.

Multidisciplinary management of hypohydrotic ectodermal dysplasia - a case report.

Hypohydrotic ectodermal dysplasia is a hereditary disorder, which affects ectodermal derivatives. It manifests several abnormalities of the teeth, and is commonly inherited through female carriers. This case report presents a patient with compromised esthetics and function. A multidisciplinary approach was planned involving an oral pathologist, endodontist, orthodontist and a prosthodontist.

Analysis of the apo(a) size polymorphism in Asian Indian populations: association with Lp(a) concentration and coronary heart disease.

Most studies aiming to detect associations of genetic variation with common complex diseases, e.g. coronary heart disease (CHD) have been performed in populations with a western lifestyle but it is unclear whether associations detected in one geographic group exist also in others. We here have determined lipoprotein(a) levels and apo(a) K-IV-2 repeat genotypes in CHD patients (N=254) and controls (N=480) from two Asian Indian populations (Tamil Nadu and New Delhi). In both populations and also in the pooled dataset median Lp(a) levels were significantly elevated in the patients (27.4 mg/dl) compared with the controls (17.6 mg/dl). Apo(a) K-IV-2 allele frequencies were not different between the CHD patients and controls and thus did not explain the increased Lp(a) levels in CHD patients. Contrary to what has recently been observed in Black and White men short (K-IVor=30) apo(a) alleles were all associated with higher Lp(a) levels in the patients. Accordingly relative risk (estimated as odds ratio) for CHD rose continuously with increasing Lp(a) but was independent of apo(a) allele length. Together with previous studies our results indicate that the relation between apo(a) genotypes, Lp(a) levels, and CHD may be heterogeneous across ethnic groups and that it depends on the genetic architecture of the Lp(a) trait in a given population whether an association of K-IV-2 repeat length with CHD exists or not.

Measurement of plasma hydroperoxide concentration by FOX-1 assay in conjunction with triphenylphosphine.

INTRODUCTION: Hydroperoxides are well-recognized reactive oxygen species which are associated with oxidative stress, a phenomenon of current clinical interest as oxidative stress is associated with a number of disease condition. Ferrous ion oxidation xylenol orange (FOX) methods of hydroperoxide estimation has outdated other methods available for hydroperoxide estimation. Two versions FOX assays are described in the literature, FOX-1 and FOX-2, in which FOX-1 is more sensitive. METHODS: we increased the sensitivity of FOX-1 assay by stabilizing the reagent pH 1.7-1.8. Analogous to FOX-2 assay, we have modified FOX-1 assay by using it in conjunction with triphenylphosphine and butylated hydroxytoluene, thus increasing the specificity of FOX-1 assay for hydroperoxide. By modified FOX-1 method, we estimated plasma hydroperoxide concentration of normal human subjects and of diabetic patients and compared with FOX-2 method. RESULTS: The FOX-1 method showed a significant high value of plasma hydroperoxide concentration compared to FOX-2 method both in normal subjects and diabetic patients with a significant correlation. By modified FOX-1 method, the recovery percentage of cumene hydroperoxide was better in biological samples when compared to FOX-2 method. CONCLUSION: The modified FOX-1 method is equally specific for hydroperoxide determination when compared to FOX-2 but is more sensitive.