Stress and Molecular Drivers for Cancer Progression: A Longstanding Hypothesis

Stress management is becoming very important part of cancer patient care. Chronic stressors lead to boost tumorigenesis and promote cancer development, recurrence, and drug resistant leading to poor health outcomes. The Hypothalamic-Pituitary-Adrenal (HPA) axis, which is activated by stress, also regulates Hypothalamic-Pituitary-Thyroid (HPT) axis. Stress related changes in immune function and inflammatory response also leads to reduced immune surveillance resulting in tumorigenesis. This article explores the hormonal axis impacted by stress and how chronic stress can lead to poor outcome of a cancer patient

Screening commercial entomopathogenic fungi for the management of Diaphorina citri populations in the Lower Rio Grande Valley, Texas, USA

Ten strains of entomopathogenic ascomycete fungi, sourced from commercial formulations of blastopore or conidiospore formulations, were tested in 14 different formulations in a primary acquisition/direct spray bioassay against adult Asian citrus psyllid (Diaphorina citri Kuwayama (Hemiptera: Liviidae)). The Cordyceps (Isaria) javanica Apopka 97-C (conidia) strain was used as the standard. A statistical ranking system was established in which top performing pathogenic strains were selected for further screening and eventual field trials. Modified Potter-type spray towers were utilized to deliver a range of doses of viable spores to adult D. citri in an aqueous spray consistent with the rate of spores per hectare often used in real-world spray applications. Mortality was assessed after a seven-day incubation period under controlled climate conditions reflecting those in the Lower Rio Grande Valley (LRGV) of Texas, USA. Of the 14 preparations, the strains Metarhizium anisopliae E9, C. fumosorosea Ifr9901, Beauveria bassiana ATCC 74040 and ANT-03, M. anisopliae ESALQ1037, and M. robertsii DWR2009, showed greater levels of mortality than the standard, Apopka 97-C, in the laboratory setting. Of those six, two (Ifr9901 and ANT-03) were selected for further evaluation based on efficacy, commercial availability, geographical registration, and market outlook on production

Plasmid Midiprep: A Method to Purify Plasmids for Recombinant DNA Studies

A fundamental aspect of molecular biology involves exploring the properties and functions of specific genes. The rise of recombinant DNA technology has vastly improved and simplified functional studies by allowing scientists to isolate specific genes using restriction enzymes and plasmids providing greater precision. Plasmids take great significance in downstream studies, which is why quantity and quality of the plasmids purified is important. In this study, we isolated and purified recombinant plasmids in microgram quantities to confirm the yield, quantity, and quality using spectral and size fractionation methods. We also assessed the plasmids for application in downstream studies. We found the plasmids that were isolated with a good yield ranging from 1-1.5 mg/ml. The high yield and purity suggest the Promega Midiprep kit is effective in producing high quality plasmids. The size of the plasmids was assessed using gel electrophoresis, and a transient transfection into mammalian cells confirmed their expression through fluorescence

Assessment of Two Novel Host-Derived Beauveria bassiana (Hypocreales: Cordycipitaceae) Isolates Against the Citrus Pest, Diaphorina citri (Hemiptera: Liviidae)

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), vectors ‘Candidatus Liberibacter spp.’, the causative agent of Citrus Greening Disease (CGD) or Huanglongbing (HLB). Managing populations of psyllids in the Lower Rio Grande Valley (LRGV), TX, United States is imperative given a continuous increase in HLB-positive trees. A component of integrated pest management (IPM) program is the use of strains of entomopathogenic fungi for the biological control of D. citri. In an attempt to find endemic strains of entomopathogenic fungi that grow favorably under LRGV environmental conditions and naturally infect D. citri, psyllids were collected from local residential areas, surface sterilized, and plated on a semi-selective agar medium. Collection of over 9,300 samples from 278 sites throughout the LRGV led to the positive identification of two Beauveria bassiana (Balsamo-Crivellii) Vuillemin (Hypocreales: Cordycipitaceae) isolates, ACP18001 and ACP18002. Chi-square analysis of primary and secondary acquisition bioassays revealed that both field isolated strains outperformed Cordyceps (Isaria) fumosorosea (Wize) (Hypocreales: Cordycipitaceae) Apopka97 under both primary (direct spray) and secondary acquisition (adult exposure to sprayed foliage) bioassays with ACP18002 marginally outperforming ACP18001 under secondary acquisition. Slopes of the dose response regression lines for the three fungi were not significantly different. In addition, the thermal profiles for vegetative growth of each isolate indicated that the field isolates grew at higher rates than the standard at higher temperatures. The new isolates may prove to be good candidates for the management of D. citri populations in the LRGV

Insights into the evolution of the New World diploid cottons (Gossypium, subgenus Houzingenia) based on genome sequencing

We employed phylogenomic methods to study molecular evolutionary processes and phylogeny in the geographically widely dispersed New World diploid cottons (Gossypium, subg. Houzingenia). Whole genome resequencing data (average of 33X genomic coverage) were generated to reassess the phylogenetic history of the subgenus and provide a temporal framework for its diversification. Phylogenetic analyses indicate that the subgenus likely originated following trans-oceanic dispersal from Africa about 6.6 mya, but that nearly all of the biodiversity evolved following rapid diversification in the mid-Pleistocene (0.5-2.0 mya), with multiple long-distance dispersals required to account for range expansion to Arizona, the Galapagos Islands, and Peru. Comparative analyses of cpDNA vs. nuclear data indicate that this history was accompanied by several clear cases of interspecific introgression. Repetitive DNAs contribute roughly half of the total 880 Mb genome, but most transposable element families are relatively old and stable among species. In the genic fraction, pairwise synonymous mutation rates average 1% per my, with non-synonymous changes being about seven times less frequent. Over 1.1 million indels were detected and phylogenetically polarized, revealing a two-fold bias toward deletions over small insertions. We suggest that this genome down-sizing bias counteracts genome size growth by TE amplification and insertions, and helps explain the relatively small genomes that are restricted to this subgenus. Compared to the rate of nucleotide substitution, the rate of indel occurrence is much lower averaging about 17 nucleotide substitutions per indel event

Insights into the Evolution of Cotton Diploids and Polyploids from Whole-Genome Re-sequencing

Understanding the composition, evolution, and function of the Gossypium hirsutum (cotton) genome is complicated by the joint presence of two genomes in its nucleus (AT and DT genomes). These two genomes were derived from progenitor A-genome and D-genome diploids involved in ancestral allopolyploidization. To better understand the allopolyploid genome, we re-sequenced the genomes of extant diploid relatives that contain the A1 (Gossypium herbaceum), A2 (Gossypium arboreum), or D5 (Gossypium raimondii) genomes. We conducted a comparative analysis using deep re-sequencing of multiple accessions of each diploid species and identified 24 million SNPs between the A-diploid and D-diploid genomes. These analyses facilitated the construction of a robust index of conserved SNPs between the A-genomes and D-genomes at all detected polymorphic loci. This index is widely applicable for read mapping efforts of other diploid and allopolyploid Gossypium accessions. Further analysis also revealed locations of putative duplications and deletions in the A-genome relative to the D-genome reference sequence. The approximately 25,400 deleted regions included more than 50% deletion of 978 genes, including many involved with starch synthesis. In the polyploid genome, we also detected 1,472 conversion events between homoeologous chromosomes, including events that overlapped 113 genes. Continued characterization of the Gossypium genomes will further enhance our ability to manipulate fiber and agronomic production of cotton

A Malvaceae mystery: A mallow maelstrom of genome multiplications and maybe misleading methods?

Previous research suggests that Gossypium has undergone a 5- to 6-fold multiplication following its divergence from Theobroma. However, the number of events, or where they occurred in the Malvaceae phylogeny remains unknown. We analyzed transcriptomic and genomic data from representatives of eight of the nine Malvaceae subfamilies. Phylogenetic analysis of nuclear data placed Dombeya (Dombeyoideae) as sister to the rest of Malvadendrina clade, but the plastid DNA tree strongly supported Durio (Helicteroideae) in this position. Intraspecific Ks plots indicated that all sampled taxa, except Theobroma (Byttnerioideae), Corchorus (Grewioideae), and Dombeya (Dombeyoideae), have experienced whole genome multiplications (WGMs). Quartet analysis suggested WGMs were shared by Malvoideae-Bombacoideae and Sterculioideae-Tilioideae, but did not resolve whether these are shared with each other or Helicteroideae (Durio). Gene tree reconciliation and Bayesian concordance analysis suggested a complex history. Alternative hypotheses are suggested, each involving two independent autotetraploid and one allopolyploid event. They differ in that one entails an allopolyploid origin for the Durio lineage, whereas the other invokes an allopolyploid origin for Malvoideae-Bombacoideae. We highlight the need for more genomic information in the Malvaceae and improved methods to resolve complex evolutionary histories that may include allopolyploidy, incomplete lineage sorting, and variable rates of gene and genome evolution

Unraveling cis and trans regulatory evolution during cotton domestication

Cis and trans regulatory divergence underlies phenotypic and evolutionary diversification. Relatively little is understood about the complexity of regulatory evolution accompanying crop domestication, particularly for polyploid plants. Here, we compare the fiber transcriptomes between wild and domesticated cotton (Gossypium hirsutum) and their reciprocal F1 hybrids, revealing genome-wide (~15%) and often compensatory cis and trans regulatory changes under divergence and domestication. The high level of trans evolution (54%–64%) observed is likely enabled by genomic redundancy following polyploidy. Our results reveal that regulatory variation is significantly associated with sequence evolution, inheritance of parental expression patterns, co-expression gene network properties, and genomic loci responsible for domestication traits. With respect to regulatory evolution, the two subgenomes of allotetraploid cotton are often uncoupled. Overall, our work underscores the complexity of regulatory evolution during fiber domestication and may facilitate new approaches for improving cotton and other polyploid plants

Anticancer drug screening using invitro Cell Proliferation assay

Introduction: In this presentation cell proliferation methods and how they are related to screening for effective chemotherapy drugs will be reviewed. Cancer in its most basic form is the unchecked mass dividing of cells while normal apoptosis is not undertaken for various reasons, some of which that have yet to be discovered. By these means’ tumors form that inhibit the functions of the organs it is residing in and the effected cells may metastasize and spread throughout the body. For this reason, chemotherapy drugs must be assessed through introduction into working strains of cultured cancer cells that are then screened for effectiveness through a process called cell proliferation assays. Objective: The goal is to find the exact dosage for inhibiting the greatest number of Colorectal Cancer Cell (CRC) cells, strain HCT-116, while leaving other healthy cells unaffected as the methods are explained. Methods: Several methods exist for determining the resulting levels of proliferation of cells after drug administration such as: Molecular Targeted Therapies (MTT) and (Water Soluble Tetrazolium) WST-1 that uses a tetrazolium salt reagent on the cells before introducing the drug in question and then a colorimetric assay is used to determine the quantity of living cells remaining through assessing which retain the dye that is produced. Alamar BLUE is another experiment that uses redox reactions but substitutes the tetrazolium with resorufin, other options include Bromodeoxyuridine (BrdU) assay which analyzes the amount of a thymidine analog that is present post experiment after it has been absorbed by denatured DNA. Results: Once cell counts have been gathered, drugs of varying concentrations have been administered, and the assays are performed to gather the number of cells that continue to proliferate, tables and graphs reflecting such information may be drawn to find the Growth Inhibitory dose of 50% (GI 50) of the cells. Discussion: At the conclusion of these trials work will still need to be done to find how these drugs will be implemented in vivo. The next logical step is moving on to testing these therapies on animals to find strengths and weaknesses in live models. Conclusion: Through the course of testing 4 different chemotherapy drugs on HCT-116, MTT and other such cell proliferation assays are utilized in finding the correct dosages to elicit the desired response of inhibiting the growth of CRC cells. The process will begin from the first splitting of cell stock to acquire workable amounts of HCT-116, culturing this working stock, and passaging it as the quantity of cells become larger. The cell proliferation methods will be outlined along with the reasoning and theory behind them to include the materials utilized. The results will be discussed while also explaining how the results are to be properly evaluated. Finally, graphed analysis resulting in either GI 50 curves may be constructed to better tabulate what the varying concentrations effects resulted in. From here we continue to narrow our search to more finite concentrations that will yield better results in killing only the exact number of cells desired