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Patchouli has begun to be widely cultivated by farmers in Burana Village with its easy cultivation and offer a promising profit. This study aims to determine whether or not it is feasible to develop a patchouli farming business in this area. Quantitative research is used to anlyze farm costs, farming profits, farm income, return cost ratio (R/C), capital productivity (n/C) and break-even point analysis (BEP). The results showed that the average area of farmers' land is 47.22 acres, so the average cost of production on patchouli farming is Rp. 23,521,073, and the average revenue obtained is Rp. 30,486,379, then the average profit for each respondent is Rp. 6,965,306, so the R/C ratio obtained is 1.3 because the value of the R/C ratio is greater than 1, so patchouli farming is feasible. The value of π/C 0.29 indicates a profitable business where each additional cost of Rp. 100 then a profit of Rp. 29 (29%). The analysis of break-even point for the production volume in patchouli farming is 53,458 kg, meaning that at 53,458 kg the patchouli business is profitable because the patchouli product number of 53,456 kg is below the average product figure of 69.998 kg. Analysis of the point of return for the cost of production in patchouli farming is Rp. 336,025 shows that it is still below the market price of Rp. 439,995, means that the patchouli business is profitable.

ABSTRACT Here, I describe a “Weekly Flow” strategy for structuring college biology courses and making organization transparent to students. Building a course around an established weekly schedule that consists of the same, or similar, elements (for example, assessments, readings, assignment types, and/or class activities) provides structure and predictability that helps students with time management and therefore enables them to engage more deeply with the course material. This flexible framework can be applied in a wide variety of course types and teaching modalities. Adding this explicit structure to a course, or simply using the framework to make existing course organization more transparent to students, is a simple intervention that supports student learning by facilitating, modeling, and providing an opportunity to practice effective time management, something many undergraduate students struggle with.

Abstract Background Histone H1 is the most mobile histone in the cell nucleus. Defining the positions of H1 on chromatin in situ, therefore, represents a challenge. Immunoprecipitation of formaldehyde-fixed and sonicated chromatin, followed by DNA sequencing (xChIP-seq), is traditionally the method for mapping histones onto DNA elements. But since sonication fragmentation precedes ChIP, there is a consequent loss of information about chromatin higher-order structure. Here, we present a new method, xxChIP-seq, employing antibody binding to fixed intact in situ chromatin, followed by extensive washing, a second fixation, sonication and immunoprecipitation. The second fixation is intended to prevent the loss of specifically bound antibody during washing and subsequent sonication and to prevent antibody shifting to epitopes revealed by the sonication process. In many respects, xxChIP-seq is comparable to immunostaining microscopy, which also involves interaction of the primary antibody with fixed and permeabilized intact cells. The only epitopes displayed after immunostaining are the “exposed” epitopes, not “hidden” by the fixation of chromatin higher-order structure. Comparison of immunoprecipitated fragments between xChIP-seq versus xxChIP-seq should indicate which epitopes become inaccessible with fixation and identify their associated DNA elements. Results We determined the genomic distribution of histone variants H1.2 and H1.5 in human myeloid leukemia cells HL-60/S4 and compared their epitope exposure by both xChIP-seq and xxChIP-seq, as well as high-resolution microscopy, illustrating the influences of preserved chromatin higher-order structure in situ. We found that xChIP and xxChIP H1 signals are in general negatively correlated, with differences being more pronounced near active regulatory regions. Among the intriguing observations, we find that transcription-related regions and histone PTMs (i.e., enhancers, promoters, CpG islands, H3K4me1, H3K4me3, H3K9ac, H3K27ac and H3K36me3) exhibit significant deficiencies (depletions) in H1.2 and H1.5 xxChIP-seq reads, compared to xChIP-seq. These observations suggest the existence of in situ transcription-related chromatin higher-order structures stabilized by formaldehyde. Conclusion Comparison of H1 xxChIP-seq to H1 xChIP-seq allows the development of hypotheses on the chromosomal localization of (stabilized) higher-order structure, indicated by the generation of “hidden” H1 epitopes following formaldehyde crosslinking. Changes in H1 epitope exposure surrounding averaged chromosomal binding sites or epigenetic modifications can also indicate whether these sites have chromatin higher-order structure. For example, comparison between averaged active or inactive promoter regions suggests that both regions can acquire stabilized higher-order structure with hidden H1 epitopes. However, the H1 xChIP-seq comparison cannot define their differences. Application of the xxChIP-seq versus H1 xChIP-seq method is particularly relevant to chromatin-associated proteins, such as linker histones, that play dynamic roles in establishing chromatin higher-order structure.

Dehydration of cells by acute hyperosmotic stress has profound effects upon cell structure and function. Interphase chromatin and mitotic chromosomes collapse (“congelation”). HL-60/S4 cells remain ~100% viable for, at least, 1 hour, exhibiting shrinkage to ~2/3 their original volume, when placed in 300mM sucrose in tissue culture medium. Fixed cells were imaged by immunostaining confocal and STED microscopy. At a “global” structural level (μm), mitotic chromosomes congeal into a residual gel with apparent (phase) separations of Ki67, CTCF, SMC2, RAD21, H1 histones and HMG proteins. At an “intermediate” level (sub-μm), radial distribution analysis of STED images revealed a most probable peak DNA density separation of ~0.16 μm, essentially unchanged by hyperosmotic stress. At a “local” structural level (~1-2 nm), in vivo crosslinking revealed essentially unchanged crosslinked products between H1, HMG and inner histones. Hyperosmotic cellular stress is discussed in terms of concepts of mitotic chromosome structure and liquid-liquid phase separation.

Tumor necrosis factor alpha (TNF-α) is a potent cytokine involved in systemic inflammation and immune modulation. Signaling responses that involve TNF-α are context dependent and capable of stimulating pathways promoting both cell death and survival. TNF-α treatment has been investigated as part of a combined therapy for acute myeloid leukemia due to its modifying effects on all-trans retinoic acid (ATRA) mediated differentiation into granulocytes. To investigate the interaction between cellular differentiation and TNF-α, we performed RNA-sequencing on two forms of the human HL-60/S4 promyelocytic leukemia cell line treated with TNF-α. The ATRA-differentiated granulocytic form of HL-60/S4 cells had an enhanced transcriptional response to TNF-α treatment compared to the undifferentiated promyelocytes. The observed TNF-α responses included differential expression of cell cycle gene sets, which were generally upregulated in TNF-α treated promyelocytes, and downregulated in TNF-α treated granulocytes. This is consistent with TNF-α induced cell cycle repression in granulocytes and cell cycle progression in promyelocytes. Moreover, we found evidence that TNF-α treatment of granulocytes shifts the transcriptome toward that of a macrophage. We conclude that TNF-α treatment promotes a divergent transcriptional program in promyelocytes and granulocytes. TNF-α promotes cell cycle associated gene expression in promyelocytes. In contrast, TNF-α stimulated granulocytes have reduced cell cycle gene expression, and a macrophage-like transcriptional program.

CENP-A histone variants replace histones H3 at centromeres. Here the authors use a single-chain antibody fragment (scFv) to stabilize human CENP-A nucleosome containing a native α-satellite DNA and solved its structure by cryo-EM to 2.6 Å resolution, providing insight into the structure and function of the CENP-A nucleosome.

Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells. VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPB), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde endoplasmic reticulum-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in endothelial cells using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted endothelial cells exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. In brief, we identified SNARE protein STX5 as a novel regulator of WPB biogenesis.

Human myeloid leukemia cells (HL-60/S4) exposed to hyperosmotic stress with sucrose undergo dehydration and cell shrinkage. Interphase chromatin and mitotic chromosomes congeal, exhibiting altered phase separation (demixing) of chromatin proteins. To investigate changes in the transcriptome, we exposed HL-60/S4 cells to hyperosmotic sucrose stress (~600 milliOsmolar) for 30 and 60 minutes. We employed RNA-Seq of polyA mRNA to identify genes with increased or decreased transcript levels relative to untreated control cells (i.e., differential gene expression). These genes were examined for over-representation of Gene Ontology (GO) terms. In stressed cells, multiple GO terms associated with transcription, translation, mitochondrial function and proteosome activity, as well as "replication-dependent histones", were over-represented among genes with increased transcript levels; whereas, genes with decreased transcript levels were over-represented with transcription repressors. The transcriptome profiles of hyperosmotically-stressed cells suggest acquisition of cellular rebuilding, a futile homeostatic response, as these cells are ultimately doomed to a dehydrated death. [ABSTRACT FROM AUTHOR]

Epigenomic regulation plays a vital role in cell differentiation. The leukemic HL-60/S4 [human myeloid leukemic cell line HL-60/S4 (ATCC CRL-3306)] promyelocytic cell can be easily differentiated from its undifferentiated promyelocyte state into neutrophil- and macrophage-like cell states. In this study, we present the underlying genome and epigenome architecture of HL-60/S4 through its differentiation. We performed whole-genome bisulphite sequencing of HL-60/S4 cells and their differentiated counterparts. With the support of karyotyping, we show that HL-60/S4 maintains a stable genome throughout differentiation. Analysis of differential Cytosine-phosphate-Guanine dinucleotide methylation reveals that most methylation changes occur in the macrophage-like state. Differential methylation of promoters was associated with immune-related terms. Key immune genes, CEBPA, GFI1, MAFB and GATA1 showed differential expression and methylation. However, we observed the strongest enrichment of methylation changes in enhancers and CTCF binding sites, implying that methylation plays a major role in large-scale transcriptional reprogramming and chromatin reorganisation during differentiation. Correlation of differential expression and distal methylation with support from chromatin capture experiments allowed us to identify putative proximal and long-range enhancers for a number of immune cell differentiation genes, including CEBPA and CCNF. Integrating expression data, we present a model of HL-60/S4 differentiation in relation to the wider scope of myeloid differentiation.

Abstract Background Mammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. The nucleus must be stiff enough to withstand cytoskeletal forces, but flexible enough to remodel as the cell changes shape. This is particularly important for cells migrating through confined spaces, where the nuclear shape must change in order to fit through a constriction. This occurs many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration. Here we characterized the effects of constricted migration in neutrophil-like cells. Results Total RNA sequencing identified that migration of neutrophil-like cells through 5- or 14-μm pores was associated with changes in the transcript levels of inflammation and chemotaxis-related genes when compared to unmigrated cells. Differentially expressed transcripts specific to migration with constriction were enriched for groups of genes associated with cytoskeletal remodeling. Hi-C was used to capture the genome organization in control and migrated cells. Limited switching was observed between the active (A) and inactive (B) compartments after migration. However, global depletion of short-range contacts was observed following migration with constriction compared to migration without constriction. Regions with disrupted contacts, TADs, and compartments were enriched for inactive chromatin. Conclusion Short-range genome organization is preferentially altered in inactive chromatin, possibly protecting transcriptionally active contacts from the disruptive effects of migration with constriction. This is consistent with current hypotheses implicating heterochromatin as the mechanoresponsive form of chromatin. Further investigation concerning the contribution of heterochromatin to stiffness, flexibility, and protection of nuclear function will be important for understanding cell migration in relation to human health and disease.

Von Willebrand factor (VWF) is a multimeric hemostatic protein that is synthesized in endothelial cells, where it is stored for secretion in elongated secretory organelles, so-called Weibel-Palade bodies (WPBs). Hemostatic activity of VWF is strongly tied to WPB length, but how endothelial cells control the dimensions of their WPBs is unclear. In this study we used a targeted shRNA screen to identify the longin-SNARE Sec22b as a novel determinant of WPB size and VWF trafficking. We found that Sec22b depletion resulted in loss of the typically elongated WPB morphology along with disintegration of the Golgi and dilation of rough ER (rER) cisternae. This was accompanied by reduced proteolytic processing of VWF, accumulation of VWF in the dilated rER and reduced basal and stimulated VWF secretion. Our data demonstrate that the elongation of WPBs, and thus adhesive activity of its cargo VWF, is determined by the rate of anterograde transport between ER and Golgi, which depends on Sec22b-containing SNARE complexes.

‘Epichromatin’, the surface of chromatin beneath the interphase nuclear envelope (NE) or at the surface of mitotic chromosomes, was discovered by immunostaining with a specific bivalent mouse monoclonal anti-nucleosome antibody (mAb PL2-6). ‘Chromomeres’, punctate chromatin particles approximately 200–300 nm in diameter, identified throughout the interphase chromatin and along mitotic chromosomes, were observed by immunostaining with the monovalent papain-derived Fab fragments of bivalent PL2-6. The specific target for PL2-6 appears to include the nucleosome acidic patch. Thus, within the epichromatin and chromomeric regions, this epitope is ‘exposed’. Considering that histones possess unstructured ‘tails’ (i.e. intrinsically disordered peptide regions, IDPR), our perception of these chromatin regions becomes more ‘fuzzy’ (less defined). We suggest that epichromatin cationic tails facilitate interactions with anionic components of NE membranes. We also suggest that the unstructured histone tails (especially, histone H1 tails), with their presumed promiscuous binding, establish multivalent binding that stabilizes each chromomere as a unit of chromatin higher order structure. We propose an ‘unstructured stability’ hypothesis, which postulates that the stability of epichromatin and chromomeres (as well as other nuclear chromatin structures) is a consequence of the collective contributions of numerous weak histone IDPR binding interactions arising from the multivalent nucleosome, analogous to antibody avidity.

Arabidopsis thaliana CELLULOSE SYNTHASE A4/7/8 (CESA4/7/8) are three non-redundant subunits of the secondary cell wall cellulose synthase complex. Transcript abundance of these genes can vary among genotypes and expression quantitative trait loci (eQTL) were identified in a recombinant population of the accessions Bay-0 and Shahdara. Genetic mapping and analysis of the transcript levels of CESAs between two distinct near isogenic lines (NILs) confirmed a change in CESA4 expression that segregates within that interval. We sequenced the promoters and identified 16 polymorphisms differentiating CESA4Sha and CESA4Bay. In order to determine which of these SNPs could be responsible for this eQTL, we screened for transcription factor protein affinity with promoter fragments of CESA4Bay, CESA4Sha, and the reference genome CESA4Col. The wall thickening activator proteins NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (NST2) and NST3 exhibited a decrease in binding with the CESA4Sha promoter with a tracheary element-regulating cis-element (TERE) polymorphism. While NILs harboring the TERE polymorphisms exhibited significantly different CESA4 expression, cellulose crystallinity and cell wall thickness were indistinguishable. These results suggest that the TERE polymorphism resulted in differential transcription factor binding and CESA4 expression; yet A. thaliana is able to tolerate this transcriptional variability without compromising the structural elements of the plant, providing insight into the elasticity of gene regulation as it pertains to cell wall biosynthesis and regulation. We also explored available DNA affinity purification sequencing data to resolve a core binding site, C(G/T)TNNNNNNNA(A/C)G, for secondary wall NACs referred to as the VNS element.

Despite years of research into microbial activity at diffuse flow hydrothermal vents, the extent of microbial niche diversity in these settings is not known. To better understand the relationship between microbial activity and the associated physical and geochemical conditions, we obtained co-registered metatranscriptomic and geochemical data from a variety of different fluid regimes within the ASHES vent field on the Juan de Fuca Ridge. Microbial activity in the majority of the cool and warm fluids sampled was dominated by a population of Gammaproteobacteria (likely sulfur oxidizers) that appear to thrive in a variety of chemically distinct fluids. Only the warmest, most hydrothermally-influenced flows were dominated by active populations of canonically vent-endemic Epsilonproteobacteria. These data suggest that the Gammaproteobacteria collected during this study may be generalists, capable of thriving over a broader range of geochemical conditions than the Epsilonproteobacteria. Notably, the apparent metabolic activity of the Gammaproteobacteria—particularly carbon fixation—in the seawater found between discrete fluid flows (the intra-field water) suggests that this area within the Axial caldera is a highly productive, and previously overlooked, habitat. By extension, our findings suggest that analogous, diffuse flow fields may be similarly productive and thus constitute a very important and underappreciated aspect of deep-sea biogeochemical cycling that is occurring at the global scale.