Subunit composition of mitochondrial dehydrogenase complexes in diplonemid flagellates.

In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a tripartite mitochondrial pyruvate dehydrogenase (PDH) complex to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes with analogous composition catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) complex and the 2-oxoglutarate dehydrogenase (OGDH) complex. Comparative transcriptome analyses of diplonemids, one of the most abundant and diverse groups of oceanic protists, indicate that the conventional E1, E2, and E3 subunits of the PDH complex are lacking. E1 was apparently replaced in the euglenozoan ancestor of diplonemids by an AceE protein of archaeal type, a substitution that we also document in dinoflagellates. Here, we demonstrate that the mitochondrion of the model diplonemid Paradiplonema papillatum displays pyruvate and 2-oxoglutarate dehydrogenase activities. Protein mass spectrometry of mitochondria reveal that the AceE protein is as abundant as the E1 subunit of BCKDH. This corroborates the view that the AceE subunit is a functional component of the PDH complex. We hypothesize that by acquiring AceE, the diplonemid ancestor not only lost the eukaryotic-type E1, but also the E2 and E3 subunits of the PDH complex, which are present in other euglenozoans. We posit that the PDH activity in diplonemids seems to be carried out by a complex, in which the AceE protein partners with the E2 and E3 subunits from BCKDH and/or OGDH.

Recent expansion of metabolic versatility in Diplonema papillatum, the model species of a highly speciose group of marine eukaryotes.

BACKGROUND: Diplonemid flagellates are among the most abundant and species-rich of known marine microeukaryotes, colonizing all habitats, depths, and geographic regions of the world ocean. However, little is known about their genomes, biology, and ecological role. RESULTS: We present the first nuclear genome sequence from a diplonemid, the type species Diplonema papillatum. The ~ 280-Mb genome assembly contains about 32,000 protein-coding genes, likely co-transcribed in groups of up to 100. Gene clusters are separated by long repetitive regions that include numerous transposable elements, which also reside within introns. Analysis of gene-family evolution reveals that the last common diplonemid ancestor underwent considerable metabolic expansion. D. papillatum-specific gains of carbohydrate-degradation capability were apparently acquired via horizontal gene transfer. The predicted breakdown of polysaccharides including pectin and xylan is at odds with reports of peptides being the predominant carbon source of this organism. Secretome analysis together with feeding experiments suggest that D. papillatum is predatory, able to degrade cell walls of live microeukaryotes, macroalgae, and water plants, not only for protoplast feeding but also for metabolizing cell-wall carbohydrates as an energy source. The analysis of environmental barcode samples shows that D. papillatum is confined to temperate coastal waters, presumably acting in bioremediation of eutrophication. CONCLUSIONS: Nuclear genome information will allow systematic functional and cell-biology studies in D. papillatum. It will also serve as a reference for the highly diverse diplonemids and provide a point of comparison for studying gene complement evolution in the sister group of Kinetoplastida, including human-pathogenic taxa.

DMA, a Small Molecule, Increases Median Survival and Reduces Radiation-Induced Xerostomia via the Activation of the ERK1/2 Pathway in Oral Squamous Cell Carcinoma.

Survival, recurrence, and xerostomia are considerable problems in the treatment of oral squamous carcinoma patients. In this study, we investigated the role of DMA (5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)5″benzimidazoyl]benzimidazole) as a salivary gland cytoprotectant in a patient-derived xenograft mouse model. A significant increase in saliva secretion was observed in the DMA-treated xenograft compared to radiation alone. Repeated doses of DMA with a high dose of radiation showed a synergistic effect on mice survival and reduced tumor growth. The mean survival rate of tumor-bearing mice was significantly enhanced. The increased number of Ki-67-stained cells in the spleen, intestine, and lungs compared to the tumor suggests DMA ablates the tumor but protects other organs. The expression of aquaporin-5 was restored in tumor-bearing mice injected with DMA before irradiation. The reduced expression of αvß3 integrin and CD44 in DMA alone and DMA with radiation-treated mice suggests a reduced migration of cells and stemness of cancer cells. DMA along with radiation treatment results in the activation of the Ras/Raf/MEK/ERK pathway in the tumor, leading to apoptosis through caspase upregulation. In conclusion, DMA has strong potential for use as an adjuvant in radiotherapy in OSCC patients.

Pixantrone confers radiosensitization in KRAS mutated cancer cells by suppression of radiation-induced prosurvival pathways.

Radioresistance towards radiation therapy has generated the need for the development of radiosensitizers as a potential drug. KRAS mutation brings radioresistance in tumor cells. The present work proves sensitization of cancer cells towards radiotherapy through inhibition of KRAS activation. Acquiring a drug repurposing approach, the in-silico screening revealed that pixantrone, an antineoplastic drug, possesses a high affinity towards KRAS G12C and G12D subtypes. The SPR study suggests that maximum affinity of pixantrone was observed with KRAS G12C>WT>G12D and G12S. Pixantrone potentially inhibited the KRAS activation in stable transfectants G12C and G12D cell lines and radiosensitized distinct KRAS mutant subtype cells. The combination of pixantrone with radiation causes enhanced dsDNA breaks along with enhanced ATM expression, and increased late apoptosis. The preclinical studies on NCr-fox1nu xenograft mice showed potent inhibition of tumor progression and prolonged survival of mcie due to the radiosensitizing effect of pixantrone. Radiation-induced activation of key effector proteins of RAS downstream pathways, like MAPK and PI3K/Akt/mTOR pathways, were downregulated in tumor cells upon combination treatment. Interestingly, a robust upregulation of senescence marker p21 was observed in the tumor cells in combination treatment. These findings reveal a convergence between KRAS signaling, pixantrone treatment, and radiation conferring tumor cell death.

Synthesis and Biological Evaluation of Novel 1H-Benzo[d]imidazole Derivatives as Potential Anticancer Agents Targeting Human Topoisomerase I.

Small molecules that modulate biological functions are targets of modern-day drug discovery efforts. A new series of novel 1H-benzo[d]imidazoles (BBZs) were designed and synthesized with different functional groups at the phenyl ring and variable lengths of the alkyl chain at the piperazine end as anticancer agents. We identified human topoisomerase I (Hu Topo I) as a probable target of these molecules through a computational study and DNA relaxation assay, a functional assay of the Hu Topo I enzyme. UV absorption, fluorescence, and circular dichroism spectroscopy were used to study interactions between BBZ and DNA. Out of 16 compounds, 11a, 12a, and 12b showed strong binding affinity and thermal stabilization of AT sequence-specific DNA. BBZs were screened against a panel of 60 human cancer cell lines at National Cancer Institute, USA. Most potent molecules 11a, 12a, and 12b showed 50% growth inhibition (GI50) in a concentration range from 0.16 to 3.6 µM cancer cells. Moreover, 12b showed 50% inhibition of the relaxation of DNA by Hu Topo I at 16 µM. Furthermore, flow cytometry revealed that 11a, 12a, and 12b cause prominent G2M arrest of cancer cells. In view of the above, we propose that 12b deserves to be further evaluated for its therapeutic use as an anticancer agent.

Molecular Detection of Streptococcus mutans and Streptococcus sobrinus in Dental Plaque Samples in Children Aged Six to Nine Years.

Context Dental caries is a widespread threat, usually in children, although it has been observed at other stages of life. Various pieces of literature have confirmed the prevalence of S treptococcus mutans and S treptococcus sobrinus in the progression of the disease. However, establishing procedures to detect these species remains a challenge, posing a barrier to treatment plans. Aim The aim of this study is to detect the species in dental plaque samples from children aged six to nine years by polymerase chain reaction (PCR) and correlate their prevalence in various dentitions. Material and Methods This is an observational analytical cross-sectional study conducted in a tertiary care dental hospital. After sample isolation, microbiological processing was performed, genomic DNA was isolated, and PCR run was performed using specific primers to detect the species. SPSS for Windows Version 17 (IBM Corp., Armonk, NY) and Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) were used to perform statistical analysis. A p-value of <0.05 was considered statistically significant. Results The technique could identify S. Mutans and S. Sobrinus in a short turnaround time. The frequency of S. mutans and S. sobrinus infections was higher in individuals with dental caries. Conclusions Molecular detection via PCR is a reliable, economical, and less time-consuming method for detecting S. mutans and S. sobrinus in dental plaque samples.

Prochlorperazine enhances radiosensitivity of non-small cell lung carcinoma by stabilizing GDP-bound mutant KRAS conformation.

Lung cancer is considered as leading cancer with the highest mortality. The KRAS-oncogenic mutations are dominant in lung carcinoma leading to poor prognosis and radioresistance, which is a major impediment to radiotherapy. Thus, KRAS mutant inhibitors that synergistically sensitize tumours to radiation are urgently needed. In pursuance of the search for a novel radiosensitizer, high-throughput screening of FDA-approved drugs was performed at active site of K-Ras. Prochlorperazine (PCZ), an antipsychotic drug, showed good binding affinity with KRAS-mutant proteins. PCZ binds to the GTP-binding pocket of KRAS-mutant protein and inhibits its constitutive activation by stabilizing the GDP-bound conformation of K-Ras mutants by 9 kcal/mol compared to WT. PCZ alongwith radiation decreased the clonogenic survival of KRAS-mutant NSCLC but not KRAS-WT cells. The combination treatment activates p-ATM, p53, and p21 proteins, leading to cell cycle arrest. PCZ with increasing radiation caused a linear increase in γH2AX foci, suggesting enhanced DSBs-associated apoptosis in radioresistant A549 cells. Pharmacokinetics study showed Cmax = 526 ng/ml at 30min, 4.6h half-life in plasma, and highest accumulation in tumours. PCZ and 10Gy irradiation synergistically radiosensitize mice xenografts via downregulation of Ras/Raf/MEK/ERK pathway. Our efforts have led to the discovery of PCZ as a lead compound. In preclinical analyses, treatment with PCZ alone and in combination with radiation led to regression of KRAS-G12S tumours.

Statins Are Associated With Increased Insulin Resistance and Secretion.

Objective: Statin treatment reduces the risk of atherosclerotic cardiovascular disease but is associated with a modest increased risk of type 2 diabetes, especially in those with insulin resistance or prediabetes. Our objective was to determine the physiological mechanism for the increased type 2 diabetes risk. Approach and Results: We conducted an open-label clinical trial of atorvastatin 40 mg daily in adults without known atherosclerotic cardiovascular disease or type 2 diabetes at baseline. The co-primary outcomes were changes at 10 weeks versus baseline in insulin resistance as assessed by steady-state plasma glucose during the insulin suppression test and insulin secretion as assessed by insulin secretion rate area under the curve (ISRAUC) during the graded-glucose infusion test. Secondary outcomes included glucose and insulin, both fasting and during oral glucose tolerance test. Of 75 participants who enrolled, 71 completed the study (median age 61 years, 37% women, 65% non-Hispanic White, median body mass index, 27.8 kg/m2). Atorvastatin reduced LDL (low-density lipoprotein)-cholesterol (median decrease 53%, P<0.001) but did not change body weight. Compared with baseline, atorvastatin increased insulin resistance (steady-state plasma glucose) by a median of 8% (P=0.01) and insulin secretion (ISRAUC) by a median of 9% (P<0.001). There were small increases in oral glucose tolerance test glucoseAUC (median increase, 0.05%; P=0.03) and fasting insulin (median increase, 7%; P=0.01). Conclusions: In individuals without type 2 diabetes, high-intensity atorvastatin for 10 weeks increases insulin resistance and insulin secretion. Over time, the risk of new-onset diabetes with statin use may increase in individuals who become more insulin resistant but are unable to maintain compensatory increases in insulin secretion.

Evaluation of Remineralizing Capacity of P11-4, CPP-ACP, Silver Diamine Fluoride, and NovaMin: An In Vitro Study.

AIM AND OBJECTIVE: To determine and compare remineralizing efficacy of NovaMin, CPP-ACP, silver diamine fluoride (SDF), and P11-4. MATERIALS AND METHODS: Sixty permanent premolars were divided into four groups with 15 samples in each group; group I: self-assembling peptide (P11-4), group II: SDF, group III: Casein phosphopeptide-stabilized amorphous calcium phosphate (CPP-ACP), and group IV: NovaMin. Mineral content was assessed using a scanning electron microscope at 7, 14, and 21 days after remineralization with each agent. RESULTS: The mean remineralization in group I at 7 days was 1.73 ± 0.02, at 14 days was 1.79 ± 0.01, and at 21 days was 1.90±0.03. Mean remineralization in group II was 1.61 ± 0.01, 1.64 ± 0.02, and 1.73 ± 0.03 at 7, 14, and 21 days, respectively. Mean remineralization in group III was 1.62 ± 0.01, 1.65 ± 0.02, and 1.74 ± 0.05 at 7, 14, and 21 days, respectively. Mean remineralization in group IV was 1.59 ± 0.02, 1.62 ± 0.07, and 1.70 ± 0.09 at 7, 14, and 21 days, respectively. The maximum value was obtained on day 21. There was a significant difference in mean remineralization values between group I vs group II, group I vs group III, and group I vs group IV (p < 0.05). CONCLUSION: Self-assembling peptides showed maximum remineralization in tested specimens followed by CPP-ACP, SDF, and NovaMin-containing toothpaste. CLINICAL SIGNIFICANCE: CPP-ACP, SDF, and NovaMin-containing toothpaste can be indicated for remineralization of initial caries in clinical use.

Treatment of Furcation Involvement Using Autogenous Tooth Graft With 1-Year Follow-Up: A Case Series.

INTRODUCTION: The objective of this study was to clinically evaluate an autogenous tooth graft (ATG) as a novel bone graft material in the treatment of Class II furcation defects. ATG is prepared at chairside from a freshly extracted tooth to be used immediately for bone regeneration. It has an advantage over the autogenous and other bone graft materials as it is non-immunogenic, inexpensive, easily available, and lacks donor-site morbidity. CASE PRESENTATION: This study was conducted on three middle-aged (35 to 55 years) male patients, who had at least one mandibular molar with Class II furcation involvement (a total of 5 sites) and one tooth that required extraction because of poor prognosis and was not endodontically treated. At 9 and 12 months, the mean reductions in horizontal probing depth were (1.40 ± 0.57 mm) and (1.52 ± 0.59 mm), respectively, and the mean gains in linear bone-fill were (3.90 ± 0.15 mm) and (5.33 ± 0.10 mm), respectively. CONCLUSIONS: Within the limitation of this study, ATG exhibited ideal properties for alveolar bone regeneration. In addition, this study outlines the chairside method to prepare a graft and highlights the improvement in clinical and radiographic parameters at 9 and 12 months.

Solution filtering affects the glassy dynamics of spincoated thin films of poly(4-chlorostyrene).

We investigated the impact of sample preparation on the glassy dynamics of thin films of poly(4-chlorostyrene), a polymer whose molecular mobility is particularly sensitive to changes in the specific volume. Samples were obtained by spincoating, the technique most commonly used to prepare thin organic layers, which consists of pouring dilute polymer solutions onto a plate rotating at a high rate. Our experimental results demonstrate that filtering the solutions before spincoating affects the value of the segmental relaxation time of the as-prepared films. Thin polymer layers obtained via filtered solutions show accelerated segmental dynamics upon confinement at the nanoscale level, once below 100nm, while the samples obtained via unfiltered solutions exhibit bulk-like dynamics down to 15-20nm. We analyzed these results by means of the cooperative free volume rate model, considering a larger free volume content in thin films obtained via filtered solutions. The validity of the model predictions was finally verified by measurements of irreversible adsorption, confirming a larger adsorbed amount, corresponding to a higher specific volume, in the case of samples obtained via unfiltered solutions. Our results prove that filtering is a crucial step in the preparation of thin films, and it could be used to switch on and off nanoconfinement effects.

Antioxidant therapy (lycopene and green tea extract) in periodontal disease: A promising paradigm.

BACKGROUND: Increased oxidative stress has emerged as one of the prime factors in the pathogenesis of periodontitis. Hence, antioxidant therapy may become a promising tool in the treatment of periodontal disease. Uric acid (UA) being a major antioxidant in saliva can be used as a marker to assess the total antioxidant capacity. AIM: The aim of the study was to investigate the influence of orally administered antioxidants (lycopene and green tea extract) on periodontal health and salivary UA levels in gingivitis patients as an adjunct to scaling and root planing (SRP). MATERIALS AND METHODS: Thirty systemically healthy participants having generalized gingivitis were randomly distributed into two groups. Control group participants received full mouth oral prophylaxis, while test group participants received oral lycopene and green tea extract (CLIK®) for 45 days along with complete oral prophylaxis. Plaque index (PI), sulcular bleeding index (SBI), and salivary UA levels were evaluated at baseline and 45 days after SRP. Data were analyzed with t-test, using SPSS software (PASW, Windows version 18.0). RESULTS: Both treatment groups demonstrated statistically highly significant (P ≤ 0.001) reduction in plaque and SBI. After treatment, a highly significant increase (P ≤ 0.001) in the test group and significant (P ≤ 0.05) increase in the control group was observed for salivary UA levels. Posttreatment comparison between test and control group delineated statistically significant results in PI (P ≤ 0.001), SBI (P ≤ 0.001), and salivary UA levels (P ≤ 0.01). CONCLUSION: Lycopene with green tea extract may prove to be a promising adjunctive prophylactic and therapeutic modality in the treatment of gingivitis patients. However, further studies are needed to evaluate the additive effect of antioxidants with routine oral prophylaxis therapy.

Comparative transcriptome analysis of shoot and root tissue of Bacopa monnieri identifies potential genes related to triterpenoid saponin biosynthesis.

BACKGROUND: Bacopa monnieri commonly known as Brahmi is utilized in Ayurveda to improve memory and many other human health benefits. Bacosides enriched standardized extract of Bacopa monnieri is being marketed as a memory enhancing agent. In spite of its well known pharmacological properties it is not much studied in terms of transcripts involved in biosynthetic pathway and its regulation that controls the secondary metabolic pathway in this plant. The aim of this study was to identify the potential transcripts and provide a framework of identified transcripts involved in bacosides production through transcriptome assembly. RESULTS: We performed comparative transcriptome analysis of shoot and root tissue of Bacopa monnieri in two independent biological replicate and obtained 22.48 million and 22.0 million high quality processed reads in shoot and root respectively. After de novo assembly and quantitative assessment total 26,412 genes got annotated in root and 18,500 genes annotated in shoot sample. Quality of raw reads was determined by using SeqQC-V2.2. Assembled sequences were annotated using BLASTX against public database such as NR or UniProt. Searching against the KEGG pathway database indicated that 37,918 unigenes from root and 35,130 unigenes from shoot were mapped to 133 KEGG pathways. Based on the DGE data we found that most of the transcript related to CYP450s and UDP-glucosyltransferases were specifically upregulated in shoot tissue as compared to root tissue. Finally, we have selected 43 transcripts related to secondary metabolism including transcription factor families which are differentially expressed in shoot and root tissues were validated by qRT-PCR and their expression level were monitored after MeJA treatment and wounding for 1, 3 and 5 h. CONCLUSIONS: This study not only represents the first de novo transcriptome analysis of Bacopa monnieri but also provides information about the identification, expression and differential tissues specific distribution of transcripts related to triterpenoid sapogenin which is one of the most important pharmacologically active secondary metabolite present in Bacopa monnieri. The identified transcripts in this study will establish a foundation for future studies related to carrying out the metabolic engineering for increasing the bacosides biosynthesis and its regulation for human health benefits.

Collective relaxation dynamics and crystallization kinetics of the amorphous Biclotymol antiseptic.

We employ dielectric spectroscopy to monitor the relaxation dynamics and crystallization kinetics of the Biclotymol antiseptic in its amorphous phase. The glass transition temperature of the material as determined by dielectric spectroscopy is Tg = 290 ± 1K. The primary (α) relaxation dynamics is observed to follow a Vogel-Fulcher-Tammann temperature dependence, with a kinetic fragility index m = 86 ± 13, which classifies Biclotymol as a relatively fragile glass former. A secondary relaxation is also observed, corresponding to an intramolecular dynamic process of the non-rigid Biclotymol molecule. The crystallization kinetics, measured at four different temperatures above the glass transition temperature, follows an Avrami behavior with exponent virtually equal to n = 2, indicating one-dimensional crystallization into needle-like crystallites, as experimentally observed, with a time-constant nucleation rate. The activation barrier for crystallization is found to be Ea = 115 ± 22 kJ mol(-1).

Antimicrobial activity of herbal medicines (tulsi extract, neem extract) and chlorhexidine against Enterococcus faecalis in Endodontics: An in vitro study.

BACKGROUND: Successful endodontic treatment depends on effective disinfection and complete sealing of root canal. Various medicaments are advised for disinfecting root canal, such as herbal and non-herbal medicaments. This study was done to assess the antimicrobial activity of herbal medicines (neem extract, tulsi extract) and chlorhexidine against Enterococcus faecalis in Endodontics. MATERIALS AND METHODS: Agar diffusion method was used to evaluate the antimicrobial action of different medicines. Sixty samples were segregated into four groups with 15 samples in each: Group I: chlorhexidine 2%, Group II: neem extract, Group III: tulsi extract, and Group IV: distilled water. The inhibition zones against E. faecalis were recorded and statistically assessed using one-way analysis of variance (ANOVA) test (P < 0.05). RESULTS: Significant antibacterial effect against E. faecalis was observed with chlorhexidine followed by neem extract and tulsi extract. CONCLUSION: Herbal medicines seemed to be effective against E. faecalis compared to 2% chlorhexidine gluconate.

Silicon-Chip-Based Dielectric Spectroscopy for Conductivity and Molecular Dynamics Studies of Organic Films.

Interdigital electrodes fabricated by standard lithography on silicon chips are employed to probe the dipolar molecular dynamics and electric conduction properties of thin rhodamine films grown with two different methods. The conductivity is due to electronic charge carriers, and at around room-temperature, it is higher by 1 order of magnitude in solution-deposited films than in thermally evaporated ones. The organic material exhibits two intrinsic dynamic processes, of which the one at higher temperature is due to the orientational motion of the dipole moment of the rhodamine units, while the one at lower temperature is due to the motion of a local dipole associated with the chlorine counterions and is absent in thermally evaporated films. Our results show that thin-film dielectric spectroscopy is an easily implementable and versatile tool to extract valuable information on thin organic films.

Sarcoidosis presenting as isolated gingival enlargement: a rare case entity.

Sarcoidosis is a non-caseating granulomatous disease . It is a multiorgan inflammatory disorder of unknown etiology. Conditions affecting skin or other organs frequently involve oral cavity and rarely manifest as gingival disease. Here we are reporting a rare case in which gingival hyperplasia was the initial symptom which finally led to the diagnosis of sarcoidosis. Oral mucous membrane needs to be examined carefully as it may constitute in presenting first sign of systemic sarcoidosis.