Mapping three-dimensional intratumor proteomic heterogeneity in uterine serous carcinoma by multiregion microsampling.

BACKGROUND: Although uterine serous carcinoma (USC) represents a small proportion of all uterine cancer cases, patients with this aggressive subtype typically have high rates of chemotherapy resistance and disease recurrence that collectively result in a disproportionately high death rate. The goal of this study was to provide a deeper view of the tumor microenvironment of this poorly characterized uterine cancer variant through multi-region microsampling and quantitative proteomics. METHODS: Tumor epithelium, tumor-involved stroma, and whole "bulk" tissue were harvested by laser microdissection (LMD) from spatially resolved levels from nine USC patient tumor specimens and underwent proteomic analysis by mass spectrometry and reverse phase protein arrays, as well as transcriptomic analysis by RNA-sequencing for one patient's tumor. RESULTS: LMD enriched cell subpopulations demonstrated varying degrees of relatedness, indicating substantial intratumor heterogeneity emphasizing the necessity for enrichment of cellular subpopulations prior to molecular analysis. Known prognostic biomarkers were quantified with stable levels in both LMD enriched tumor and stroma, which were shown to be highly variable in bulk tissue. These USC data were further used in a comparative analysis with a data generated from another serous gynecologic malignancy, high grade serous ovarian carcinoma, and have been added to our publicly available data analysis tool, the Heterogeneity Analysis Portal ( https://lmdomics.org/ ). CONCLUSIONS: Here we identified extensive three-dimensional heterogeneity within the USC tumor microenvironment, with disease-relevant biomarkers present in both the tumor and the stroma. These data underscore the critical need for upfront enrichment of cellular subpopulations from tissue specimens for spatial proteogenomic analysis.

E-cadherin Expression in Premalignant Lesions, Premalignant Conditions, Oral Squamous Cell Carcinoma, and Normal Mucosa: An Immunohistochemical Study.

Background Oral squamous cell carcinoma (OSCC) is a multi-step process. Epithelial-mesenchymal transition (EMT) is an important step in the progression of OSCC. One of the components that influence EMT is E-cadherin. The aim of this study was to determine the expression of E-cadherin in oral submucous fibrosis (OSMF), various grades of epithelial dysplasia, OSCC, and to compare it with the expression in the normal mucosa. Material and methods E-cadherin immunohistochemical detection was done using a monoclonal antibody of clone EP-6TM and the PolyExcel HRP/DAB chromogen detection system. A total of 100 samples, were divided into four groups, which included epithelial dysplasia (group 2) (30 cases), oral submucous fibrosis (group 3) (OSMF-30 cases), and oral squamous cell carcinoma (group 4) (OSCC-30 cases), which was compared with normal mucosa (group 1) (10 cases). The positive control used for E-cadherin was ductal breast carcinoma. Results All the cases of normal mucosa, epithelial dysplasia, and OSMF showed positivity for E-cadherin expression. In OSCC, 97% of cases expressed E-cadherin except one case. Out of 30 cases of epithelial dysplasia, 53% of mild epithelial dysplasia had a moderate intensity of expression and 75% had a mild intensity of E-cadherin expression. In moderately differentiated OSCC, 82% of cases showed mild intensity. Tissue localization of the E-cadherin stain in the basal layer decreased from normal mucosa to grades of epithelial dysplasia and OSCC. The pattern of E-cadherin staining in all the cases of group I, group II, and group III was membranous. In 97% of OSCC cases, both membranous and cytoplasmic staining were seen. Conclusion E-cadherin expression was reduced in increasing grades of epithelial dysplasia, OSCC, and OSMF compared to that of normal mucosa. E-cadherin expression is reduced as the lesions progress to malignancy. Hence, E-cadherin can be considered a surrogate marker of malignancy.

Host-delivered RNAi-mediated silencing using fusion cassettes of different functional groups of genes precludes Meloidogyne incognita multiplication in Nicotiana tabacum.

KEY MESSAGE: This study demonstrates multi-gene silencing approach for simultaneous silencing of several functional genes through a fusion gene strategy for protecting plants against root-knot nematode, Meloidogyne incognita. The ability of root-knot nematode (RKN), Meloidogyne incognita, to cause extensive yield decline in a wide range of cultivated crops is well-documented. Due to the inadequacies of current management approaches, the alternatively employed contemporary RNA interference (RNAi)-based host-delivered gene silencing (HD-RNAi) strategy targeting different functional effectors/genes has shown substantial potential to combat RKNs. In this direction, we have explored the possibility of simultaneous silencing of four esophageal gland genes, six plant cell-wall modifying enzymes (PCWMEs) and a serine protease gene of M. incognita using the fusion approach. In vitro RNAi showed that combinatorial gene silencing is the most effective in affecting nematode behavior in terms of reduced attraction, penetration, development, and reproduction in tomato and adzuki beans. In addition, qRT-PCR analysis of M. incognita J2s soaked in fusion-dsRNA showed perturbed expression of all the genes comprising the fusion construct confirming successful dsRNA processing which is also supported by increased mRNA abundance of five key-RNAi pathway genes. In addition, hairpin RNA expressing constructs of multi-gene fusion cassettes were developed and used for generation of Nicotiana tabacum transgenic plants. The integration of gene constructs and expression of siRNAs in transgenic events were confirmed by Southern and Northern blot analyses. Besides, bio-efficacy analyses of transgenic events, conferred up to 87% reduction in M. incognita multiplication. Correspondingly, reduced transcript accumulation of the target genes in the M. incognita females extracted from transgenic events confirmed successful gene silencing.

Patient-reported tolerability of adjuvant ipilimumab (3 or 10 mg/kg) versus high-dose interferon alfa-2b for resected high-risk stage III-IV melanoma in phase III trial E1609.

PURPOSE: Trial E1609 demonstrated superior overall survival with ipilimumab 3 mg/kg (ipi3) compared to high-dose interferon (HDI) for patients with resected high-risk melanoma. To inform treatment tolerability, we compared health-related quality of life (HRQoL), gastrointestinal (GI), and treatment-specific physical and cognitive/emotional symptoms. We also compared treatment-specific concerns between all arms. METHODS: We assessed HRQoL using the Functional Assessment of Cancer Therapy-General, physical and cognitive/emotional concerns using the FACT-Biologic Response Modifier subscale, and GI symptoms with the Functional Assessment of Chronic Illness Therapy-Diarrhea subscale pre-treatment and every 3 months. The primary outcome was the difference in HRQoL at 3 months between ipi3/ipi10 vs. HDI. RESULTS: 549 patients (n = 158 ipi3; n = 191 ipi10; n = 200 HDI) were analyzed. 3-month completion was 58.7%. Compared to HDI, ipilimumab patients reported better HRQoL (ipi3 = 87.5 ± 14.6 vs. HDI = 74.7 ± 15.4, p < .001; ipi10 = 84.9 ± 16.5 vs. HDI, p < .001) and fewer physical (ipi3 = 22.3 ± 4.6 vs. HDI = 17.1 ± 5.4, p < .001; ipi10 = 21.8 ± 5.0 vs. HDI p < .001) and cognitive/emotional (ipi3 = 18.6 ± 4.4 vs. HDI = 15.0 ± 5.3, p < .001; ipi10 = 17.7 ± 4.8 vs. HDI p < .001) concerns, but worse GI symptoms (ipi3 = 40.8 ± 5.0 vs. HDI = 42.2 ± 2.9, p = .011; ipi10 = 39.5 ± 7.0 vs. HDI, p < .001). Fewer ipilimumab patients reported worsening treatment-specific concerns (e.g., 52% of ipi3 and 58% of ipi10 reported worsening fatigue vs. 82% HDI, p's < .001). CONCLUSION: PROs demonstrated less toxicity of ipi3 compared to HDI and ipi10. Priorities for symptom management among patients receiving ipilimumab include GI toxicities, fatigue, weakness, appetite loss, arthralgia, and depression. TRIAL REGISTRATION: NCT01274338, January 11, 2011 (first posted date) https://clinicaltrials.gov/ct2/show/NCT01274338?term=NCT01274338&draw=2&rank=1 .

Amenability of Maruca vitrata (Lepidoptera: Crambidae) to gene silencing through exogenous administration and host-delivered dsRNA in pigeonpea (Cajanus cajan L.).

Insect pests are one of the major biotic stresses limiting yield in commercially important crops. The lepidopteran polyphagous spotted pod borer, Maruca vitrata causes significant economic losses in legumes including pigeonpea. RNA interference (RNAi)-based gene silencing has emerged as one of the potential biotechnological tools for crop improvement. We report in this paper, RNAi in M. vitrata through exogenous administration of dsRNA with sequence specificity to three functionally important genes, Alpha-amylase (α-amylase), Chymotrypsin-like serine protease (CTLP) and Tropomyosin (TPM) into the larval haemolymph and their host-delivered RNAi in pigeonpea. Significant decline in the expression of selected genes supported by over-expression of DICER and generation of siRNA indicated the occurrence of RNAi in the dsRNA-injected larvae. Additionally, the onset of RNAi in the herbivore was demonstrated in pigeonpea, one of the prominent hosts, by host-delivered dsRNA. Transgenics in pigeonpea (cv. Pusa 992), a highly recalcitrant crop, were developed through a shoot apical meristem-targeted in planta transformation strategy and evaluated. Plant level bioassays in transgenic events characterized and selected at molecular level showed mortality of M. vitrata larvae as well as reduced feeding when compared to wild-type. Furthermore, molecular evidence for down regulation of target genes in the insects that fed on transgenic plants authenticated RNAi. Considering the variability of gene silencing in lepidopteran pests, this study provided corroborative proof for the possibility of gene silencing in M. vitrata through both the strategies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-022-01133-3.

Social determinants of trauma care: Associations of race, insurance status, and place on opioid prescriptions, postdischarge referrals, and mortality.

BACKGROUND: Racial disparities in trauma care have been reported for a range of outcomes, but the extent to which these remain after accounting for socioeconomic and environmental factors remains unclear. The objective of this study was to evaluate the unique contributions of race, health insurance, community distress, and rurality/urbanicity on trauma outcomes after carefully controlling for specific injury-related risk factors. METHODS: All adult (age, ≥18 years) trauma patients admitted to a single Level I trauma center with a statewide, largely rural, catchment area from January 2010 to December 2020 were retrospectively reviewed. Primary outcomes were mortality, rehabilitation referral, and receipt of opioids in the emergency department. Demographic, socioeconomic, and injury characteristics as well as indicators of community distress and rurality based on home address were abstracted from a trauma registry database. RESULTS: Analyses revealed that Black patients (n = 13,073) were younger, more likely to be male, more likely to suffer penetrating injuries, and more likely to suffer assault-based injuries compared with White patients (n = 10,946; all p < 0.001). In adjusted analysis, insured patients had a 28% lower risk of mortality (odds ratio, 0.72; p = 0.005) and were 92% more likely to be referred for postdischarge rehabilitation than uninsured patients (odds ratio, 1.92; p = 0.005). Neither race- nor place-based factors were associated with mortality. However, post hoc analyses revealed a significant race by age interaction, with Black patients exhibiting more pronounced increases in mortality risk with increasing age. CONCLUSION: The present findings help disentangle the social determinants of trauma disparities by adjusting for place and person characteristics. Uninsured patients were more likely to die and those who survived were less likely to receive referrals for rehabilitation services. The expected racial disparity in mortality risk favoring White patients emerged in middle age and was more pronounced for older patients. LEVEL OF EVIDENCE: Prognostic and epidemiological, Level III.

Deciphering the Behavioral Response of Meloidogyne incognita and Fusarium oxysporum Toward Mustard Essential Oil.

Environmental concerns related to synthetic pesticides and the emphasis on the adoption of an integrated pest management concept as a cardinal principle have strengthened the focus of global research and development on botanical pesticides. A scientific understanding of the mode of action of biomolecules over a range of pests is key to the successful development of biopesticides. The present investigation focuses on the in silico protein-ligand interactions of allyl isothiocyanate (AITC), a major constituent of black mustard (Brassica nigra) essential oil (MEO) against two pests, namely, Meloidogyne incognita (Mi) and Fusarium oxysporum f. sp. lycopersici (Fol), that cause severe yield losses in agricultural crops, especially in vegetables. The in vitro bioassay results of MEO against Mi exhibited an exposure time dependent on the lethal concentration causing 50% mortality (LC50) values of 47.7, 30.3, and 20.4 µg ml-1 at 24, 48, and 72 h of exposure, respectively. The study revealed short-term nematostatic activity at lower concentrations, with nematicidal activity at higher concentrations upon prolonged exposure. Black mustard essential oil displayed excellent in vitro Fol mycelial growth inhibition, with an effective concentration to cause 50% inhibition (EC50) value of 6.42 µg ml-1. In order to decipher the mechanism of action of MEO, its major component, AITC (87.6%), which was identified by gas chromatography-mass spectrometry (GC-MS), was subjected to in silico docking and simulation studies against seven and eight putative target proteins of Mi and Fol, respectively. Allyl isothiocyanate exhibited the highest binding affinity with the binding sites of acetyl cholinesterase (AChE), followed by odorant response gene-1 (ODR1) and neuropeptide G-protein coupled receptor (nGPCR) in Mi, suggesting the possible suppression of neurotransmission and chemosensing functions. Among the target proteins of Fol, AITC was the most effective protein in blocking chitin synthase (CS), followed by 2,3-dihydroxy benzoic acid decarboxylase (6m53) and trypsinase (1try), thus inferring these as the principal molecular targets of fungal growth. Taken together, the study establishes the potential of MEO as a novel biopesticide lead, which will be utilized further to manage the Mi-Fol disease complex.

Host-mediated RNAi for simultaneous silencing of different functional groups of genes in Meloidogyne incognita using fusion cassettes in Nicotiana tabacum.

KEY MESSAGE: This study establishes possibility of combinatorial silencing of more than one functional gene for their efficacy against root-knot nematode, M. incognita. Root-knot nematodes (RKN) of the genus Meloidogyne are the key important plant parasitic nematodes (PPNs) in agricultural and horticultural crops worldwide. Among RKNs, M. incognita is the most notorious that demand exploration of novel strategies for their management. Due to its sustainable and target-specific nature, RNA interference (RNAi) has gained unprecedented importance to combat RKNs. However, based on the available genomic information and interaction studies, it can be presumed that RKNs are dynamic and not dependent on single genes for accomplishing a particular function. Therefore, it becomes extremely important to consider silencing of more than one gene to establish any synergistic or additive effect on nematode parasitism. In this direction, we have combined three effectors specific to subventral gland cells of M. incognita, Mi-msp1, Mi-msp16, Mi-msp20 as fusion cassettes-1 and two FMRFamide-like peptides, Mi-flp14, Mi-flp18, and Mi-msp20 as fusion cassettes-2 to establish their possible utility for M. incognita management. In vitro RNAi assay in tomato and adzuki bean using these two fusion gene negatively altered nematode behavior in terms of reduced attraction, invasion, development, and reproduction. Subsequently, Nicotiana tabacum plants were transformed with these two fusion gene hairpin RNA-expressing vectors (hpRNA), and characterized via PCR, qRT-PCR, and Southern blot hybridization. Production of siRNAs specific to Mi-flp18 and Mi-msp1 was also confirmed by Northern hybridization. Further, transgenic events expressing single copy insertions of hpRNA constructs of fusion 1 and fusion-2 conferred up to 85% reduction in M. incognita multiplication. Besides, expression quantification revealed a significant reduction in mRNA abundance of target genes (up to 1.8-fold) in M. incognita females extracted from transgenic plants, and provided additional evidence for successful gene silencing.

Extensive three-dimensional intratumor proteomic heterogeneity revealed by multiregion sampling in high-grade serous ovarian tumor specimens.

Enriched tumor epithelium, tumor-associated stroma, and whole tissue were collected by laser microdissection from thin sections across spatially separated levels of ten high-grade serous ovarian carcinomas (HGSOCs) and analyzed by mass spectrometry, reverse phase protein arrays, and RNA sequencing. Unsupervised analyses of protein abundance data revealed independent clustering of an enriched stroma and enriched tumor epithelium, with whole tumor tissue clustering driven by overall tumor "purity." Comparing these data to previously defined prognostic HGSOC molecular subtypes revealed protein and transcript expression from tumor epithelium correlated with the differentiated subtype, whereas stromal proteins (and transcripts) correlated with the mesenchymal subtype. Protein and transcript abundance in the tumor epithelium and stroma exhibited decreased correlation in samples collected just hundreds of microns apart. These data reveal substantial tumor microenvironment protein heterogeneity that directly bears on prognostic signatures, biomarker discovery, and cancer pathophysiology and underscore the need to enrich cellular subpopulations for expression profiling.

Rapid and sensitive detection of potato cyst nematode Globodera rostochiensis by loop-mediated isothermal amplification assay.

Cyst nematodes of the species Globodera rostochiensis and G. pallida are devastating parasites of the potato crop. Early detection of cyst nematodes in the field is critical for adopting an appropriate management strategy. A specific and sensitive loop-mediated isothermal amplification (LAMP) assay using four oligonucleotide primers has been developed to amplify the internal transcribed spacer region (ITS) of ribosomal DNA of potato cyst nematode G. rostochiensis. The PCN-LAMP reaction could be completed within 75 min at 68 °C followed by termination at 85 °C for 7 min. The primers exhibited specificity for G. rostochiensis and did not detect any other tested genera of plant parasitic or entomopathogenic nematodes. LAMP reaction was highly sensitive, suitable for crude genomic DNA and could successfully detect G. rostochiensis DNA up to femtogram quantity. This assay is rapid, cost effective and requires minimal instrumentation. It will facilitate the detection of G. rostochiensis at field and point-of-care labs and help in the interception of infested plant material/soil samples at quarantine stations independent of a professional nematologist. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02830-8.

Exogenous administration of dsRNA for the demonstration of RNAi in Maruca vitrata (lepidoptera: crambidae).

The polyphagous spotted pod borer, Maruca vitrata is an important agricultural pest that causes extensive damage on various food crops. Though the pest is managed by synthetic chemicals, exploration of biotechnological approaches for its control is important. RNAi-based gene silencing is one such tool that has been extensively used for functional genomics and is highly variable in insects. In view of this, we have attempted to demonstrate RNAi in M. vitrata through exogenous double-stranded RNA (dsRNA) administration targeting seven genes associated with midgut, chemosensory, cell signalling and development. Two modes of exogenous dsRNA delivery by either haemolymph injection and/or ingestion into third and late third instar larval stages respectively exhibited efficient silencing of specific transcripts. Furthermore, dsRNA injection into the haemolymph showed significant reduction of target gene expression compared to negative controls establishing this mode of delivery to be more efficient. Interestingly, haemolymph injection required lesser dsRNA and led to higher reduction of transcript level vis-à-vis ingestion as demonstrated in dsRNA Serine Protease 33 (ds-SP33)-fed larvae. Over-expression of key RNAi component DICER and detection of siRNA authenticated the presence of RNAi in M. vitrata. Additionally, we have identified inhibitor molecules like morpholine, piperidine, carboxamide and piperidine-carboxamide through in silico analysis for blocking the function of SP33 to demonstrate the utility of functional genomics. Thus, the present study establishes the usefulness of injection and ingestion approaches for exogenous dsRNA delivery into M. vitrata larvae for effective RNAi. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02741-8.

Micro RNA-induced gene silencing strategy for the delivery of siRNAs targeting Meloidogyne incognita in a model plant Nicotiana benthamiana.

BACKGROUND: Occurrence of multiple biotic stresses on crop plants result in drastic yield losses which may have severe impact on the food security. It is a challenge to design strategies for simultaneous management of these multiple stresses. Hence, establishment of innovative approaches that aid in their management is critical. Here, we have introgressed a micro RNA-induced gene silencing (MIGS) based combinatorial gene construct containing seven target gene sequences of cotton leaf curl disease (CLCuD), cotton leaf hopper (Amrasca biguttula biguttula), cotton whitefly (Bemisia tabaci) and root-knot nematode (Meloidogyne incognita). RESULTS: Stable transgenic lines of Nicotiana benthamiana were generated with the T-DNA harboring Arabidopsis miR173 target site fused to fragments of Sec23 and ecdysone receptor (EcR) genes of cotton leaf hopper and cotton whitefly. It also contained C2/replication associated protein (C2/Rep) and C4 (movement protein) along with ßC1 gene of betasatellite to target CLCuD, and two FMRFamide-like peptide (FLP) genes, Mi-flp14 and Mi-flp18 of M. incognita. These transgenic plants were assessed for the amenability of MIGS approach for pest control by efficacy evaluation against M. incognita. Results showed successful production of small interfering RNA (siRNA) through the tasiRNA (trans-acting siRNA) pathway in the transgenic plants corresponding to Mi-flp18 gene. Furthermore, we observed reduced Mi-flp14 and Mi-flp18 transcripts (up to 2.37 ± 0.12-fold) in females extracted from transgenic plants. The average number of galls, total endoparasites, egg masses and number of eggs per egg mass reduced were in the range 27-62%, 39-70%, 38-65% and 34-49%, respectively. More importantly, MIGS transgenic plants showed 80% reduction in the nematode multiplication factor (MF). CONCLUSION: This study demonstrates successful validation of the MIGS approach in the model plant, N. benthamiana for efficacy against M. incognita, as a prelude to translation to cotton. © 2021 Society of Chemical Industry.

Characterization and in planta validation of a CHI4 chitinase from Cajanus platycarpus (Benth.) Maesen for its efficacy against pod borer, Helicoverpa armigera (Hübner).

BACKGROUND: Pigeonpea, Cajanus cajan is one of the economically important legume food crops and a major source of dietary proteins. Management of pod borer, Helicoverpa armigera has been prominent among crop improvement programs. Lack of resistance sources in the cultivated germplasm and crossing incompatibility with pod borer-resistant wild relatives have prompted biotechnological interventions. Identification and exploitation of genes from pigeonpea wild relatives in host plant resistance towards the pod borer assumes pertinence. Dynamic transcriptome analysis of the wild relative vis a vis cultivated pigeonpea identified a CHI4 chitinase as one of the putative insect resistance genes. RESULTS: The study presents variations in important amino acids in CHI4 chitinases from C. cajan and its wild relative C. platycarpus. Comparative protein modeling and docking analysis of the two proteins demonstrated differences in substrate binding efficacy of the chitinase from C. platycarpus which resulted in a minimum binding energy of -8.7 kcal mol-1 . Furthermore, we successfully evaluated the insecticidal activity of the chitinase from C. platycarpus against H. armigera challenge through heterologous expression in tobacco. Molecular characterization of transgenic plants confirmed that their efficacy against H. armigera was a result of the integration of CHI4 from C. platycarpus. CONCLUSION: Docking analysis demonstrated effective substrate interaction as a possible reason for efficacy against pod borer in the chitinase from C. platycarpus. This was authenticated by successful overexpression and bioefficacy assessment against H. armigera in tobacco. The CHI4 gene from C. platycarpus can be useful in the mitigation of H. armegira in pigeonpea as well as in other crops. © 2021 Society of Chemical Industry.

Simultaneous RNAi Knockdown of Three FMRFamide-Like Peptide Genes, Mi-flp1, Mi-flp12, and Mi-flp18 Provides Resistance to Root-Knot Nematode, Meloidogyne incognita.

Root-knot nematode, Meloidogyne incognita, is a devastating sedentary endoparasite that causes considerable damage to agricultural crops worldwide. Modern approaches targeting the physiological processes have confirmed the potential of FMRFamide like peptide (FLPs) family of neuromotor genes for nematode management. Here, we assessed the knock down effect of Mi-flp1, Mi-flp12, and Mi-flp18 of M. incognita and their combinatorial fusion cassette on infection and reproduction. Comparative developmental profiling revealed higher expression of all three FLPs in the infective 2nd stage juveniles (J2s). Further, Mi-flp1 expression in J2s could be localized in the ventral pharyngeal nerves near to metacarpal bulb of the central nervous system. In vitro RNAi silencing of three FLPs and their fusion cassette in M. incognita J2s showed that combinatorial silencing is the most effective and affected nematode host recognition followed by reduced penetration ability and subsequent infection into tomato and adzuki bean roots. Northern blot analysis of J2s soaked in fusion dsRNA revealed the presence of siRNA of all three target FLPs establishing successful processing of fusion gene dsRNA in the J2s. Further, evaluation of the fusion gene cassette is done through host-delivered RNAi in tobacco. Transgenic plants with fusion gene RNA-expressing vector were generated in which transgene integration was confirmed by PCR, qRT-PCR, and Southern blot analysis. Transcript accumulation of three FLPs constituting the fusion gene was reduced in the M. incognita females collected from the transgenic plants that provided additional evidence for successful gene silencing. Evaluation of positive T1 transgenic lines against M. incognita brought down the disease burden as indicated by various disease parameters that ultimately reduced the nematode multiplication factor (MF) by 85% compared to the wild-type plants. The study establishes the possibility of simultaneous silencing of more than one FLPs gene for effective management of M. incognita.

Expression interference of a number of Heterodera avenae conserved genes perturbs nematode parasitic success in Triticum aestivum.

The cereal cyst nematode, Heterodera avenae is distributed worldwide and causes substantial damage in bread wheat, Triticum aestivum. This nematode is extremely difficult to manage because of its prolonged persistence as unhatched eggs encased in cysts. Due to its sustainable and target-specific nature, RNA interference (RNAi)-based strategy has gained unprecedented importance for pest control. To date, RNAi strategy has not been exploited to manage H. avenae in wheat. In the present study, 40 H. avenae target genes with different molecular function were rationally selected for in vitro soaking analysis in order to assess their susceptibility to RNAi. In contrast to target-specific downregulation of 18 genes, 7 genes were upregulated and 15 genes showed unaltered expression (although combinatorial soaking showed some of these genes are RNAi susceptible), suggesting that a few of the target genes were refractory or recalcitrant to RNAi. However, RNAi of 37 of these genes negatively altered nematode behavior in terms of reduced penetration, development and reproduction in wheat. Subsequently, wheat plants were transformed with seven H. avenae target genes (that showed greatest abrogation of nematode parasitic success) for host-induced gene silencing (HIGS) analysis. Transformed plants were molecularly characterized by PCR, RT-qPCR and Southern hybridization. Production of target gene-specific double- and single-stranded RNA (dsRNA/siRNA) was detected in transformed plants. Transgenic expression of galectin, cathepsin L, vap1, serpin, flp12, RanBPM and chitinase genes conferred 33.24-72.4 % reduction in H. avenae multiplication in T1 events with single copy ones exhibiting greatest reduction. A similar degree of resistance observed in T2 plants indicated the consistent HIGS effect in the subsequent generations. Intriguingly, cysts isolated from RNAi plants were of smaller size with translucent cuticle compared to normal size, dark brown control cysts, suggesting H. avenae developmental retardation due to HIGS. Our study reinforces the potential of HIGS to manage nematode problems in crop plant.

Impact of adjuvant treatment and prognostic factors in stage I uterine leiomyosarcoma patients treated in Commission on Cancer®-accredited facilities.

OBJECTIVES: Determine the impact of adjuvant chemotherapy (ACT) and prognostic factors in surgically managed patients with stage I uterine leiomyosarcoma (ULMS). METHODS: Women who underwent hysterectomy and were diagnosed with stage I ULMS between 2010 and 2014 in the National Cancer Database were eligible for this observation study. Inverse probability of treatment weighting based on propensity score was used to balance clinical characteristics between ACT and no ACT patients. Hazard ratio (HR) and 95% confidence interval (CI) were estimated from Cox modeling. RESULTS: There were 1059 eligible patients with stage I ULMS including 514 treated with ACT and 545 with no ACT. Patient characteristics and tumor features varied in patients treated with ACT vs. no ACT (P < .0001). Multivariate survival analysis demonstrated that patient age, comorbidity score, tumor size, lymphovascular space invasion (LVSI) and grade were independent prognostic factors. After propensity score weighting to control for imbalance of prognostic clinical factors, adjusted five-year survival was 61.7% vs. 61.3% and restricted mean survival time was 39.7 vs. 40.6 months for ACT vs. no ACT, respectively. Risk of death in a weighted Cox analysis of overall survival was similar (HR = 1.08, 95% CI = 0.85-1.37, P = .054) for ACT vs. no ACT patients. Subset analysis demonstrated that survival was similar in ACT vs. no ACT patients categorized by age, tumor size and LVSI or with high grade or ungraded tumors. In contrast, patients with low grade tumors had worse 5-year survival (82.3% vs. 91.5%) and an increased risk of death (HR = 3.79, 95% CI = 1.15-12.40, P = .028) following ACT vs. no ACT. CONCLUSIONS: ACT did not improve survival over no ACT in patients with stage I ULMS and was inferior in patients with low grade tumors.

Pathology of Melanoma.

Melanoma is an aggressive malignancy arising from melanocytes in the skin and rarely in extracutaneous sites. The understanding of pathology of melanoma has evolved over the years, with the initial classifications based on the clinical and microscopic features to the current use of immunohistochemistry and genetic sequencing. The depth of invasion and lymph node metastasis are still the most important prognostic features of melanoma. Other important prognostic features include ulceration, lymphovascular invasion, mitosis, and tumor-infiltrating lymphocytes. This article reviews the pathology of melanoma and its precursor lesions, along with the recent advances in pathologic diagnosis of melanoma.

Phase III Study of Adjuvant Ipilimumab (3 or 10 mg/kg) Versus High-Dose Interferon Alfa-2b for Resected High-Risk Melanoma: North American Intergroup E1609.

PURPOSE: Phase III adjuvant trials have reported significant benefits in both relapse-free survival (RFS) and overall survival (OS) for high-dose interferon alfa (HDI) and ipilimumab at 10 mg/kg (ipi10). E1609 evaluated the safety and efficacy of ipilimumab at 3 mg/kg (ipi3) and ipi10 versus HDI. PATIENTS AND METHODS: E1609 was a phase III trial in patients with resected cutaneous melanoma (American Joint Committee on Cancer 7th edition stage IIIB, IIIC, M1a, or M1b). It had 2 coprimary end points: OS and RFS. A 2-step hierarchic approach first evaluated ipi3 versus HDI followed by ipi10 versus HDI. RESULTS: Between May 2011 and August 2014, 1,670 adult patients were centrally randomly assigned (1:1:1) to ipi3 (n = 523), HDI (n = 636), or ipi10 (n = 511). Treatment-related adverse events grade ≥ 3 occurred in 37% of patients receiving ipi3, 79% receiving HDI, and 58% receiving ipi10, with adverse events leading to treatment discontinuation in 35%, 20%, and 54%, respectively. Comparison of ipi3 versus HDI used an intent-to-treat analysis of concurrently randomly assigned patient cases (n = 1,051) and showed significant OS difference in favor of ipi3 (hazard ratio [HR], 0.78; 95.6% repeated CI, 0.61 to 0.99; P = .044; RFS: HR, 0.85; 99.4% CI, 0.66 to 1.09; P = .065). In the second step, for ipi10 versus HDI (n = 989), trends in favor of ipi10 did not achieve statistical significance. Salvage patterns after melanoma relapse showed significantly higher rates of ipilimumab and ipilimumab/anti-programmed death 1 use in the HDI arm versus ipi3 and ipi10 (P ≤ .001). CONCLUSION: Adjuvant therapy with ipi3 benefits survival versus HDI; for the first time to our knowledge in melanoma adjuvant therapy, E1609 has demonstrated a significant improvement in OS against an active control regimen. The currently approved adjuvant ipilimumab dose (ipi10) was more toxic and not superior in efficacy to HDI.

Meloidogyne incognita (Nematoda: Meloidogynidae) sterol-binding protein Mi-SBP-1 as a target for its management.

Meloidogyne incognita is a polyphagous plant-parasitic nematode that causes considerable yield loss in agricultural and horticultural crops. The management options available for M. incognita are extremely limited. Here we identified and characterised a M. incognita homolog of Caenorhabditis elegans sterol-binding protein (Mi-SBP-1), a transcriptional regulator of several lipogenesis pathway genes, and used RNA interference-mediated gene silencing to establish its utility as a target for the management of M. incognita. Mi-sbp-1 is predicted to be a helix-loop-helix domain containing DNA binding transcription factor, and is present in the M. incognita genome in three copies. The RNA-Seq analysis of Mi-sbp-1 silenced second stage juveniles confirmed the key role of this gene in lipogenesis regulation in M. incognita. In vitro and host-induced gene silencing of Mi-sbp-1 in M. incognita second stage juveniles resulted in loss of nematodes' ability to utilise the stored fat reserves, slower nematode development, and reduced parasitism on adzuki bean and tobacco plants. The multiplication factor for the Mi-sbp-1 silenced nematodes on adzuki bean plants was reduced by 51% compared with the control nematodes in which Mi-sbp-1 was not silenced. Transgenic expression of the double-stranded RNA construct of the Mi-sbp-1 gene in tobacco plants caused 40-45% reduction in M. incognita multiplication, 30-43.8% reduction in the number of egg masses, and 33-54% reduction in the number of eggs per egg mass compared with the wild type control plants. Our results confirm that Mi-sbp-1 is a key regulator of lipogenesis in M. incognita and suggest that it can be used as an effective target for its management. The findings of this study can be extended to develop methods to manage other economically important parasitic nematodes.

Evaluating and comparing the morphological and histopathological changes induced by erbium:yttrium-aluminum-garnet laser and diamond bur on enamel, dentin and pulp tissue.

AIM: Lasers are used for different types of dental treatments. Using the erbium:yttrium-aluminum-garnet (Er:YAG) laser to remove dental hard tissue is simple, advantageous and influences the type of cavity preparation, whether conventional or conservative in nature. The aim of the present study was to evaluate and compare the morphological and histopathological changes in the enamel, dentin and pulp tissue of the teeth treated by Er:YAG laser and conventional burs. METHODS: A conventional class I cavity was prepared in orthodontic patients by laser and bur. The teeth were extracted and analyzed for morphological changes using a scanning electron microscope, ground sections and histopathological changes under a light microscope. RESULTS: The time with laser was longer than the conventional methods. The lased cavity showed irregular appearance with absence of smear layer which is suitable for the resin restoration. The ground section and the histopathological study showed no differences between the groups. CONCLUSION: The Er:YAG laser is effective in the removal of dental hard tissue without damaging the pulp when coupled with ideal energy output. It is widely used in different dental fields. It needs time to be accepted by dentist and patients and further studies are required to explore its advantages.