Microscale Gene Expression Analysis of Tumor-Associated Macrophages

Macrophages, apart from being the key effector cells of the innate immune system, also play critical roles during the development and progression of various complex diseases, including cancer. Tumor-associated macrophages, infiltrate tumors during different stages of cancer progression to regulate motility, invasion, and intravasation to metastatic sites. Macrophages can exist in different polarization states associated with unique function in tumors. Since tumor-associated macrophages constitute a very small proportion of tumor cells, analysis of gene expression pattern using normal extraction buffer-based methods remains a challenging task. Therefore, it is imperative to develop low-throughput strategies to investigate transcriptional regulations from a small number of immune cells. Here, we describe an efficient, sensitive, and cost-effective approach for gene expression analysis of a small number of fluorescence-activated sorted tumor-associated macrophages. Our analyses from the different number of stable, primary, and sorted macrophages suggest 5,000 cells is an optimal number for performing quantitative, real-time PCR analysis of multiple genes. Our studies could detect expression of macrophage-specific genes from cultured primary macrophages, and FACS-sorted macrophages from different biological tissues without introducing biases in comparative gene expression ratios. In conclusion, our kit-based method for quantitative gene expression analysis from a small number of cells found in biological tissues will provide an opportunity to study cell-specific, transcriptional changes

Poboljšanje potisnute imunoreaktivnosti prouzročene virusom zarazne anemije pilića upotrebom imunomodulatora i hematika.

Nutritional immunomodulation represents a new frontier area of research for achieving rational goals of efficient and healthy livestock production, as continuous genetic selection and intensive productions systems have made the animals highly susceptible to various pathogens. In this study, the amelioration of haematinic and immunomodulator on chicken infectious anemia virus (CIAV) induced immmunosupression was studied, with regard to haematological and immunological parameters. Vaccines against Newcastle disease (ND) and infectious bursal disease (IBD) were used as markers for immunological parameter studies. A total of 80 specific pathogen free (SPF) broiler chicks, negative for CIAV antibodies, were randomly divided into four groups (Group I-IV) and were vaccinated against ND and IBD on days 1 and 12, respectively. All chicks of groups II, II and IV were inoculated with CIAV intramuscularly on day 15 of the study, while group I served as control. The chicks of group III and IV were treated with a haematinic (haemocare) and haematinic (haemocare) + immunomodulator (Immuno care), respectively, in drinking water for the entire study period (28 days). Live body weights, haematological parameters, along with humoral immune response (HIR) were measured at regular intervals. The CIAV infected groups showed a significant decline in cell count of erythrocyte and most leukocyte lineages, reduction in live body weights, and antibody titres against ND and IBD viruses. Theintensity of body weight loss, anaemia, leukopenia and depressed humoral immune responses were less severe in CIAV infected chicks fed with haematenic and immunomodulator supplementation diets. Interestingly, dually treated chicks (group IV) had a significantly higher response than the group treated by haematinic alone . In conclusion, the use of immunomodulator plus haematinic supplementation is suggested for providing better protection compared to haematinic supplementation alone, and the combination can be used for prophylaxis and therapeutic purposes against CIA, an economically important and emerging disease of poultry.Imunomodulacija putem hrane predstavlja novo područje istraživanja s ciljem da se postigne učinkovita proizvodnja zdrave stoke, jer su trajni genetski odabir i zahtjevni proizvodni sustavi učinili životinje visoko prijemljivima na različite patogene. U ovom je radu, na osnovi hematoloških i imunoloških pokazatelja, istražen učinak hematika i imunomodulatora na imunosupresiju izazvanu virusom zarazne anemije pilića. Cjepiva protiv newcastleske bolesti i zaraznog burzitisa kokoši rabljena su kao osnova za istraživanje imunoloških pokazatelja. Ukupno je 80 tovnih pilića, slobodnih od specifičnih patogenih uzročnika, negativnih na protutijela za virus zarazne anemije pilića, bilo nasumce podijeljeno u četiri skupine (I-IV) te prvog dana cijepljeno protiv newcastleske bolesti, a 12. dana protiv zaraznog burzitisa kokoši. Svi pilići skupine II, III i IV bili su intramuskularno zaraženi virusom zarazne anemije pilića 15. dana pokusa, dok je skupina I bila kontrolna. Pilići skupine III dobivali su hematik, a skupine IV hematik i imunomodulator u pitkoj vodi tijekom čitavog razdoblja istraživanja (28 dana). Tjelesna masa, hematološki pokazatelji i humoralni imunosni odgovor bili su mjereni u pravilnim razmacima. Skupina zaražena virusom zarazne anemije pokazivala je značajni pad broja eritrocita i leukocita te smanjenje tjelesne mase i titra protutijela za virus newcastleske bolesti i virus zaraznog burzitisa. Gubitak tjelesne mase, anemija, leukopenija i humoralni imunosni odgovor bili su slabiji u pilića zaraženih virusom zarazne anemije koji su dobivali hematik i imunomodulator u hrani. Zanimljivo je da je u pilića koji su dobivali dvostruki dodatak (skupina IV) zabilježen značajno bolji odgovor nego u pilića koji su dobivali samo hematik. Može se zaključiti da je upotreba imunomodulatora i hematika pružila bolju zaštitu u odnosu na upotrebu samog hematika. Kombinacija se može upotrijebiti za profilaksu i terapiju protiv zarazne anemije pilića, gospodarski važne i emergentne zarazne bolesti

Foliar feeding of boron influencing biochemical attributes and enzyme activity in dragon fruit (Selenicereus monacanthus)

Boron plays crucial role in metabolic processes during fruit ripening and in turn ensures better fruit quality. However limited studies have been conducted to assess the influence of boron on fruit quality of dragon fruit. In the present study, the efficacy of boron was investigated on red-fleshed dragon fruit (Selenicereus monacanthus). Four levels of boron (100 mgL-1, 200 mgL-1, 300 mgL-1 and 400 mgL-1) were applied on 7- and 14-day-old flower buds. The highest pollen germinability, seed weight, fruit weight (274.32 ± 36.72g), pulp content (70.80 ± 1.79%) and pulp firmness (2.74 ± 0.18 N) were recorded when B was applied@300 mg L-1 on 7-day old flower bud. The same treatment also manifested higher soluble solid contents (17.42 ± 0.62 °Brix), sugar content, total carbohydrate (15.92 ± 1.12%), protein (1.33±0.11%), ascorbic acid (112.66 ± 4.98 µg/g), betacyanin (32.86±2.52 µg/g), total phenol (95.26 ± 3.72 µg GAE/ 100g), total flavonoid (37.65 ±2.14 mg QE/100g) and anti-oxidative activity (27.71±2.14 mM Fe II/100g). Correlation studies elucidated significant positive influence of pollen germinability on fruit weight, pulp content and pulp firmness. The activities of α-amylase, invertase and sucrose synthase enzymes were significantly upregulated with the application of B 300 mg L-1 on 7-day old flower bud. On the other hand, the activities of cell wall degrading enzymes such as cellulase, polygalacturonase and pectin methyl esterase were reduced with increasing levels of boron. The principal component analysis (PCA) illustrated the maximal proximity of most of the quality attributes with B 300 mgL-1, applied at 7-day old flower bud stage, thus exemplifying it as the best treatment

Metabolic Rewiring by Loss of Sirt5 Promotes Kras-induced Pancreatic Cancer Progression.

BACKGROUND & AIMS: SIRT5 plays pleiotropic roles via post-translational modifications, serving as a tumor suppressor, or an oncogene, in different tumors. However, the role SIRT5 plays in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) remains unknown. METHODS: Published datasets and tissue arrays with SIRT5 staining were used to investigate the clinical relevance of SIRT5 in PDAC. Furthermore, to define the role of SIRT5 in the carcinogenesis of PDAC, we generated autochthonous mouse models with conditional Sirt5 knockout. Moreover, to examine the mechanistic role of SIRT5 in PDAC carcinogenesis, SIRT5 was knocked down in PDAC cell lines and organoids, followed by metabolomics and proteomics studies. A novel SIRT5 activator was utilized for therapeutic studies in organoids and patient-derived xenografts. RESULTS: SIRT5 expression negatively regulated tumor cell proliferation and correlated with a favorable prognosis in PDAC patients. Genetic ablation of Sirt5 in PDAC mouse models promoted acinar-to-ductal metaplasia, precursor lesions, and pancreatic tumorigenesis, resulting in poor survival. Mechanistically, SIRT5 loss enhanced glutamine and glutathione metabolism via acetylation-mediated activation of GOT1. A selective SIRT5 activator, MC3138, phenocopied the effects of SIRT5 overexpression and exhibited anti-tumor effects on human PDAC cells. MC3138 also diminished nucleotide pools, sensitizing human PDAC cell lines, organoids, and patient-derived xenografts to gemcitabine. CONCLUSIONS: Collectively, we identify SIRT5 as a key tumor suppressor in PDAC, whose loss promotes tumorigenesis through increased non-canonical utilization of glutamine via GOT1, and that SIRT5 activation is a novel therapeutic strategy to target PDAC

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Not AvailableScreening of thirty-two allozyme loci in Tor putitora yielded ten polymorphic loci, useful in determining genetic divergence in natural populations of the species. Samples from three different Indian rivers viz. the Satluj, Ganga and Garua were studied with the identified allozyme loci, to assess their suitability for genetic diversity analysis in T. putitora. The observed heterozygosity per locus varied from 0.0840 (Satluj) to 0.1245 (Ganga). The significant genotype heterogeneity was observed between the sample sets (P<0.05). The identified loci exhibited significant potential to determine population structure of T. putitora across its natural range of distribution.Not Availabl

Influence of Some Plant Growth Regulators on Quality Attributes of Pineapple (Ananas comosus L.)

Aims: A trial was conducted to investigate the influence of plant growth regulators on the growth and quality attributes of pineapple fruits. Study Design: The experiment was arranged in a randomized completed block design (RBCD) with three replications using 25 plants per replication. Place and Duration of Study: The experiment was carried out between 2020 and 2021 at the Central Horticultural Experiment Station, Aignia, Bhubaneswar, India. Methodology: The application of PGRs i.e. naphthalene acetic acid (NAA), gibberellic acid (GA), and brassinosteroid (BR) was done at the flower initiation stage. Only water application is done in case of control. Fruits were harvested at the ripe stage and an analysis of different growth and quality parameters were done. Results: The application of naphthalene acetic acid (NAA), and gibberellic acid (GA) significantly improved the yield and quality attributes of pineapple. Application of NAA 200 mgL-1 (T3) was found to be the most effective in enhancing absolute growth rate (2.65 g day-1), fruit weight (1.58 kg), and fruit/crown ratio (6.38). It was also evident that the application of NAA 200 mgL-1 improved fruit quality in terms of soluble solid contents (17.67 oBrix), soluble solid contents to acid ratio (19.49), reducing sugar (3.54%), and total sugar content (12.38%). Higher pulp content (73.89%), pulp-to-peel ratio (3.49), and juice content (54.50%) were also observed with NAA 200 mgL-1. Conclusion: NAA 200 mgL-1 significantly improved fruit growth i.e. fruit weight, fruit-to-crown ratio, and absolute growth rate. It also enhanced the quality attributes of pineapple fruits i.e. pulp content, pulp-to-peel ratio, juice content, pH, soluble solid contents, SSC to acid ratio, reducing sugar, and total sugar content