Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.

PNPLA3 Genotype and Dietary Fat Modify Concentrations of Plasma and Fecal Short Chain Fatty Acids and Plasma Branched-Chain Amino Acids.

The GG genotype of the Patatin-like phosphatase domain-containing 3 (PNPLA3), dietary fat, short-chain fatty acids (SCFA) and branched-chain amino acids (BCAA) are linked with non-alcoholic fatty liver disease. We studied the impact of the quality of dietary fat on plasma (p) and fecal (f) SCFA and p-BCAA in men homozygous for the PNPLA3 rs738409 variant (I148M). Eighty-eight randomly assigned men (age 67.8 ± 4.3 years, body mass index 27.1 ± 2.5 kg/m2) participated in a 12-week diet intervention. The recommended diet (RD) group followed the National and Nordic nutrition recommendations for fat intake. The average diet (AD) group followed the average fat intake in Finland. The intervention resulted in a decrease in total p-SCFAs and iso-butyric acid in the RD group (p = 0.041 and p = 0.002). Valeric acid (p-VA) increased in participants with the GG genotype regardless of the diet (RD, 3.6 ± 0.6 to 7.0 ± 0.6 µmol/g, p = 0.005 and AD, 3.8 ± 0.3 to 9.7 ± 8.5 µmol/g, p = 0.015). Also, genotype relation to p-VA was seen statistically significantly in the RD group (CC: 3.7 ± 0.4 to 4.2 ± 1.7 µmol/g and GG: 3.6 ± 0.6 to 7.0 ± 0.6 µmol/g, p = 0.0026 for time and p = 0.004 for time and genotype). P-VA, unlike any other SCFA, correlated positively with plasma gamma-glutamyl transferase (r = 0.240, p = 0.025). Total p-BCAAs concentration changed in the AD group comparing PNPLA3 CC and GG genotypes (CC: 612 ± 184 to 532 ± 149 µmol/g and GG: 587 ± 182 to 590 ± 130 µmol/g, p = 0.015 for time). Valine decreased in the RD group (p = 0.009), and leucine decreased in the AD group (p = 0.043). RD decreased total fecal SCFA, acetic acid (f-AA), and butyric acid (f-BA) in those with CC genotype (p = 0.006, 0.013 and 0.005, respectively). Our results suggest that the PNPLA3 genotype modifies the effect of dietary fat modification for p-VA, total f-SCFA, f-AA and f-BA, and total p-BCAA.

Gut ecological networks reveal associations between bacteria, exercise, and clinical profile in non-alcoholic fatty liver disease patients.

IMPORTANCE: Our study is applying a community-based approach to examine the influence of exercise on gut microbiota (GM) and discover GM structures linked with NAFLD improvements during exercise. The majority of microbiome research has focused on finding specific species that may contribute to the development of human diseases. However, we believe that complex diseases, such as NAFLD, would be more efficiently treated using consortia of species, given that bacterial functionality is based not only on its own genetic information but also on the interaction with other microorganisms. Our results revealed that exercise significantly changes the GM interaction and that structural alterations can be linked with improvements in intrahepatic lipid content and metabolic functions. We believe that the identification of these characteristics in the GM enhances the development of exercise treatment for NAFLD and will attract general interest in this field.

Pectin from sunflower by-products obtained by ultrasound: Chemical characterization and in vivo evaluation of properties in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a public health challenge and the use of pectin for symptom amelioration is a promising option. In this work, sunflower pectin has been extracted without (CHP) and with assistance of ultrasound (USP) using sodium citrate as a food-grade extracting agent. At optimal conditions (64 °C, 23 min) the highest yield was obtained with ultrasound application (15.5 vs. 8.1 %). Both pectins were structurally characterized by 1H NMR, HPSEC-ELSD, FT-IR and GC-FID. Unlike CHP, USP showed a lower molecular weight, higher galacturonic acid, lower degree of methyl-esterification and, overall, higher viscosity. These characteristics could affect the anti-inflammatory activity of pectins, evaluated using DSS-induced IBD model mice. So, USP promoted the defence (ICAM-1) and repair of the gastrointestinal mucosa (TFF3, ZO-1) more effectively than CHP. These results demonstrate the potential amelioration of acute colitis in IBD mice through USP supplementation. Taking into account the biomarkers analysed, these results demonstrate, for the first time, the positive impact of sunflower pectin extracted by ultrasound under very soft conditions on inflammatory bowel disease that might open up new possibilities in the treatment of this serious pathology.

Twelve Weeks of High-Intensity Interval Training Alters Adipose Tissue Gene Expression but Not Oxylipin Levels in People with Non-Alcoholic Fatty Liver Disease.

Lifestyle modifications, including increased physical activity and exercise, are recommended for non-alcoholic fatty liver disease (NAFLD). Inflamed adipose tissue (AT) contributes to the progression and development of NAFLD and oxylipins such as hydroxyeicosatetraenoic acids (HETE), hydroxydocosahexanenoic acids (HDHA), prostaglandins (PEG2), and isoprostanoids (IsoP), which all may play a role in AT homeostasis and inflammation. To investigate the role of exercise without weight loss on AT and plasma oxylipin concentrations in NAFLD subjects, we conducted a 12-week randomized controlled exercise intervention. Plasma samples from 39 subjects and abdominal subcutaneous AT biopsy samples from 19 subjects were collected both at the beginning and the end of the exercise intervention. In the AT of women, a significant reduction of gene expression of hemoglobin subunits (HBB, HBA1, HBA2) was observed within the intervention group during the 12-week intervention. Their expression levels were negatively associated with VO2max and maxW. In addition, pathways involved in adipocyte morphology alterations significantly increased, whereas pathways in fat metabolism, branched-chain amino acids degradation, and oxidative phosphorylation were suppressed in the intervention group (p < 0.05). Compared to the control group, in the intervention group, the ribosome pathway was activated, but lysosome, oxidative phosphorylation, and pathways of AT modification were suppressed (p < 0.05). Most of the oxylipins (HETE, HDHA, PEG2, and IsoP) in plasma did not change during the intervention compared to the control group. 15-F2t-IsoP significantly increased in the intervention group compared to the control group (p = 0.014). However, this oxylipin could not be detected in all samples. Exercise intervention without weight loss may influence the AT morphology and fat metabolism at the gene expression level in female NAFLD subjects.

Immobilization of Alcalase on Silica Supports Modified with Carbosilane and PAMAM Dendrimers.

Enzyme immobilization is a powerful strategy for enzyme stabilization and recyclability. Materials covered with multipoint molecules are very attractive for this goal, since the number of active moieties to attach the enzyme increases with respect to monofunctional linkers. This work evaluates different dendrimers supported on silica to immobilize a protease enzyme, Alcalase. Five different dendrimers were employed: two carbosilane (CBS) dendrimers of different generations (SiO2-G0Si-NH2 and SiO2-G1Si-NH2), a CBS dendrimer with a polyphenoxo core (SiO2-G1O3-NH2), and two commercial polyamidoamine (PAMAM) dendrimers of different generations (SiO2-G0PAMAM-NH2 and SiO2-G1PAMAM-NH2). The results were compared with a silica support modified with a monofunctional molecule (2-aminoethanethiol). The effect of the dendrimer generation, the immobilization conditions (immobilization time, Alcalase/SiO2 ratio, and presence of Ca2+ ions), and the digestion conditions (temperature, time, amount of support, and stirring speed) on Alcalase activity has been evaluated. Enzyme immobilization and its activity were highly affected by the kind of dendrimer and its generation, observing the most favorable behavior with SiO2-G0PAMAM-NH2. The enzyme immobilized on this support was used in two consecutive digestions and, unlike CBS supports, it did not retain peptides released in the digestion.

Effects of exercise on NAFLD using non-targeted metabolomics in adipose tissue, plasma, urine, and stool.

The mechanisms by which exercise benefits patients with non-alcoholic fatty liver disease (NAFLD), the most common liver disease worldwide, remain poorly understood. A non-targeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics analysis was used to identify metabolic changes associated with NAFLD in humans upon exercise intervention (without diet change) across four different sample types-adipose tissue (AT), plasma, urine, and stool. Altogether, 46 subjects with NAFLD participated in this randomized controlled intervention study. The intervention group (n = 21) performed high-intensity interval training (HIIT) for 12 weeks while the control group (n = 25) kept their sedentary lifestyle. The participants' clinical parameters and metabolic profiles were compared between baseline and endpoint. HIIT significantly decreased fasting plasma glucose concentration (p = 0.027) and waist circumference (p = 0.028); and increased maximum oxygen consumption rate and maximum achieved workload (p < 0.001). HIIT resulted in sample-type-specific metabolite changes, including accumulation of amino acids and their derivatives in AT and plasma, while decreasing in urine and stool. Moreover, many of the metabolite level changes especially in the AT were correlated with the clinical parameters monitored during the intervention. In addition, certain lipids increased in plasma and decreased in the stool. Glyco-conjugated bile acids decreased in AT and urine. The 12-week HIIT exercise intervention has beneficial ameliorating effects in NAFLD subjects on a whole-body level, even without dietary changes and weight loss. The metabolomics analysis applied to the four different sample matrices provided an overall view on several metabolic pathways that had tissue-type specific changes after HIIT intervention in subjects with NAFLD. The results highlight especially the role of AT in responding to the HIIT challenge, and suggest that altered amino acid metabolism in AT might play a critical role in e.g. improving fasting plasma glucose concentration.Trial registration ClinicalTrials.gov (NCT03995056).

A Promotor-Led Pilot Study to Increase Colorectal Cancer Screening in Latinos: The Juntos Contra El Cáncer Program.

Background. Latinos have lower colorectal cancer (CRC) screening rates compared to other racial/ethnic groups in the United States, despite an overall increase in CRC screening over the past 10 years. To address this disparity, we implemented a promotor-led intervention to increase CRC screening test adherence in community-based settings, connecting community members with a partnering federally qualified health center. Purpose. To evaluate the Juntos Contra el Cáncer/Together Against Cancer (JUNTOS) intervention, by assessing pre-post changes in (1) CRC screening test adherence and (2) CRC knowledge and perceived barriers to CRC screening. We also assessed the feasibility and acceptability of program activities. Method. JUNTOS was a group-based intervention, delivered by promotores (community health workers), to promote CRC screening test adherence among Latino adults. The intervention consisted of a culturally tailored 2½-hour interactive workshop followed by an appointment scheduling assistance from a promotor. Workshop participants were Latino adults (males and females) aged 50 to 75 years who were not up-to-date with CRC screening guidelines. We conducted interviews before and 6 to 9 months after the workshop to assess program outcomes. Results. Of the 177 participants included, 118 reported completing the CRC screening test (66.7%) by 6 to 9 months postintervention. We observed baseline to 6- to 9-month increase in CRC knowledge and lower perceived barriers to obtaining CRC screening. Furthermore, the intervention was found to be feasible and acceptable. Conclusion. Results suggest that JUNTOS can be feasibly implemented in partnership with a federally qualified health center. The current study supports group-based CRC interventions in community and clinic settings.

Immobilization of thermolysin enzyme on dendronized silica supports. Evaluation of its feasibility on multiple protein hydrolysis cycles.

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles.

Functionalization of silica with amine and ammonium alkyl chains, dendrons and dendrimers: Synthesis and antibacterial properties.

Materials modified with ammonium groups on the surface have shown antibacterial activity. In this paper, alkyl chains, carbosilane (CBS) dendrimers and dendrons and poly(amidoamine) (PAMAM) dendrimers containing amine and ammonium groups have been grafted to silica surface and the influence of molecule structure on the stability and on antibacterial activity have been evaluated. These materials have been characterized by thermogravimetric analysis (TGA), zeta (Z) potential, scanning electron microscopy (SEM), infrared spectroscopy (IR) and nuclear magnetic resonance (13C CP MAS NMR). The degree of silica functionalization depends on type of outer groups, amine or ammonium, type and core of dendrimer, and length of chains. The Z potential measurements of these materials in water suspensions were used to test their stability in this medium. These measurements showed, for some of the modified silicas, the diminishing of Z potential from positive values toward zero, probably due to interaction of the functional groups with the silica surface. This variation was also dependent on ligand structure and peripheral functions. Finally, studies of inhibition of bacteria growth stand out again the relevance of ligand structure and number of functional groups on silica surface. The most active systems were those with more surface covered, those with cationic groups further away from silica surface and higher dendritic generation.

Anticancer Activity of Dendriplexes against Advanced Prostate Cancer from Protumoral Peptides and Cationic Carbosilane Dendrimers.

The interaction of neuropeptides, vasoactive intestinal peptide (VIP), or growth hormone-releasing hormone (GHRH), with a cationic carbosilane dendrimer forms dendriplexes with antitumoral behavior in advanced prostate cancer cells PC3. At the concentrations used for dendriplexes formation, the free peptides were protumoral and prometastatic in advanced prostate cancer, while dendrimer only showed low cytotoxicity, but did not avoid the metastatic behavior of PC3 cells. However, these nanoplexes favored also cell adhesion and avoided cell migration. Also, the dendriplexes were not toxic for no tumoral prostate cells (RPWE-1) or fibroblasts. The use of labeled GHRH peptide (rhodamine labeled) and a dendrimer (fluorescein labeled) allowed us to observe that both systems reach the intracellular milieu after dendriplex formation. The treatment of PC3 cells with the nanoplexes reduced expression of vascular endothelial growth factor (VEGF) and cyclic adenosine monophosphate (cAMP). Molecular modeling analysis highlights the important contribution of the carbosilane framework in the stabilization of the dendriplex, since dendrimer interacts with a peptide region where hydrophobic amino acids are presented.

Serum, liver and bile sitosterol and sitostanol in obese patients with and without NAFLD.

Background and aims: Non-alcoholic fatty liver disease (NAFLD) associates with low levels of serum plant sterols in cross-sectional studies. In addition, it has been suggested that the hepatic sterol transport mechanisms are altered in NAFLD. Therefore, we investigated the association between serum, liver and bile plant sterols and sitostanol with NAFLD.Methods: Out of the 138 individuals (age: 46.3 ± 8.9, body mass index: 43.3 ± 6.9 kg/m², 28% men and 72% women), 44 could be histologically categorized to have normal liver, and 94 to have NAFLD. Within the NAFLD group, 28 had simple steatosis and 27 had non-alcoholic steatohepatitis. Plant sterols and sitostanol were measured from serum (n=138), liver (n=38), and bile (n=41). The mRNA expression of genes regulating liver sterol metabolism and inflammation was measured (n=102).Results: Liver and bile sitostanol ratios to cholesterol were higher in those with NAFLD compared to those with histologically normal liver (all P<0.022). Furthermore, liver sitostanol to cholesterol ratio correlated positively with histological steatosis and lobular inflammation (rs > 0.407, P<0.01 for both). In contrast, liver sitosterol to cholesterol ratio correlated negatively with steatosis (rs = -0.392, P=0.015) and lobular inflammation (rs = -0.395, P=0.014). Transcriptomics analysis revealed suggestive correlations between serum plant sterol levels and mRNA expression.Conclusion: Our study showed that liver and bile sitostanol ratios to cholesterol associated positively and liver sitosterol ratio to cholesterol associated negatively with liver steatosis and inflammation in obese individuals with NAFLD..

Study of non-covalent interactions on dendriplex formation: Influence of hydrophobic, electrostatic and hydrogen bonds interactions.

The interaction of a double stranded small interference RNA (siRNA Nef) with cationic carbosilane dendrimers of generations 1-3 with two different ammonium functions at the periphery ([-NMe2R]+, R=Me, (CH2)2OH) has been studied by experimental techniques (zeta potential, electrophoresis, single molecule pulling experiments) and molecular dynamic calculations. These studies state the presence of different forces on dendriplex formation, depending on generation and type of ammonium group. Whilst for higher dendrimers electrostatic forces mainly drive the stability of dendriplexes, first generation compounds can penetrate into siRNA strands due to the establishment of hydrophobic interactions. Finally, in the particular case of first generation dendrimer [G1O3(NMe2(CH2)2OH))6]6+; the presence of hydroxyl groups reinforces dendriplex stability by hydrogen bonds formation. However, since these small dendrimers do not cover the RNA, only higher generation derivatives protect RNA from degradation.

Serum Plant Sterols Associate with Gallstone Disease Independent of Weight Loss and Non-Alcoholic Fatty Liver Disease.

BACKGROUND: Gallstone disease (GD) has been associated with low serum levels of plant sterols. We evaluated the impact of laparoscopic Roux-en-Y gastric bypass (LRYGB) and non-alcoholic fatty liver disease (NAFLD) on the association of GD with low levels of serum plant sterols. METHODS: Two hundred forty-two consecutive morbidly obese patients were recruited to this prospective study. Histological analysis of liver biopsy to diagnose NAFLD was performed. Bile sample was taken during the LRYGB. Associations of GD with serum non-cholesterol sterol to cholesterol ratios, measured using gas liquid chromatography and with mRNA expression of genes participating in the cholesterol, bile, and fatty acid metabolism in the liver, were analyzed. RESULTS: Out of the 242 participants, 95 had GD. Lower weight (p = 0.002) and female sex (p = 0.0006) were associated with GD. Serum plant sterols, campesterol (p = 0.003), sitosterol (p = 0.002), and avenasterol (p = 0.015), were lower in patients with GD compared to those without GD. This association remained significant after adjustment for NAFLD, use of statin medication, and previous laparoscopic cholecystectomy (LCC). Levels of sitosterol (p = 0.001) and campesterol (p = 0.001) remained lower in obese individuals with GD also after obesity surgery. Liver mRNA expression of genes regulating cholesterol synthesis and bile metabolism was increased in individuals with GD. CONCLUSIONS: Serum plant sterols were lower in patients with GD independent of NAFLD, history of LCC, use of statin medication, and weight loss after LRYGB. Low serum plant sterols in patients with GD suggest potentially inherited alterations in sterol absorption and biliary transport in subjects susceptible for GD.

Bio-identity and fate of albumin-coated SPIONs evaluated in cells and by the C. elegans model.

UNLABELLED: Nanoparticles which surface adsorb proteins in an uncontrolled and non-reproducible manner will have limited uses as nanomedicinal products. A promising approach to avoid nanoparticle non-specific interactions with proteins is to design bio-hybrids by purposely pre-forming a protein corona around the inorganic cores. Here, we investigate, in vitro and in vivo, the newly acquired bio-identity of superparamagnetic iron oxide nanoparticles (SPIONs) upon their functionalization with a pre-formed and well-defined bovine serum albumin (BSA) corona. Cellular uptake, intracellular particle distribution and cytotoxicity were studied in two cell lines: adherent and non-adherent cells. BSA decreases nanoparticle internalization in both cell lines and protects the iron core once they have been internalized. The physiological response to the nanoparticles is then in vivo evaluated by oral administration to Caenorhabditis elegans, which was selected as a model of a functional intestinal barrier. Nanoparticle biodistribution, at single particle resolution, is studied by transmission electron microscopy. The analysis reveals that the acidic intestinal environment partially digests uncoated SPIONs but does not affect BSA-coated ones. It also discloses that some particles could enter the nematode's enterocytes, likely by endocytosis which is a different pathway than the one described for the worm nutrients. STATEMENT OF SIGNIFICANCE: Unravelling meaningful relationships between the physiological impact of engineered nanoparticles and their synthetic and biological identity is of vital importance when considering nanoparticles biomedical uses and when establishing their nanotoxicological profile. This study contributes to better comprehend the inorganic nanoparticles' behavior in real biological milieus. We synthesized a controlled pre-formed BSA protein corona on SPIONs to lower unspecific cell uptake and decrease nanoparticle fouling with other proteins. Such findings may be of relevance considering clinical translation and regulatory issues of inorganic nanoparticles. Moreover, we have advanced in the validation of C. elegans as a simple animal model for assessing biological responses of engineering nanomaterials. The physiological response of BSA coated SPIONs was evaluated in vivo after their oral administration to C. elegans. Analyzing ultra-thin cross-sections of the worms by TEM with single-particle precision, we could track NP biodistribution along the digestive tract and determine unambiguously their translocation through biological barriers and cell membranes.

[Cronkhite-Canada syndrome - a rare intestinal polypotic syndrome]. / Cronkhite-Canadan oireyhtymä.

Cronkhite-Canada syndrome is a rare polypotic disease of the gastrointestinal tract. There are no specific diagnostic criteria for this syndrome. Suspicion may arise if both an endoscopic and a histopathologic finding together with the patient's symptom picture are indicative of the disease. Other polypotic conditions should be excluded. No established treatment is available for the syndrome. The principles of treatment have remained almost unchanged at least over the past decade. With a large proportion of the patients eventually developing a gastrointestinal malignancy, the prognosis of the disease is mostly poor.

Transfer of carboplatin and paclitaxel into breast milk.

Carboplatin is an alkylating agent that is FDA approved for the treatment of advanced ovarian cancer. Paclitaxel is a plant taxane mitotic inhibitor approved for primary or salvage treatment of ovarian and breast cancer. This is a case report of a 40-year-old woman who was exclusively breastfeeding prior to being treated for papillary thyroid cancer with intravenous carboplatin (233 mg) and intravenous paclitaxel (30 mg/m(2)) for 6 consecutive weeks. Breast milk samples were collected during the sixth chemotherapy session. Carboplatin had a relative infant dose of 2.0% and remained measurable after 316 hours. Paclitaxel had a relative infant dose of 16.7% but was eliminated before 316 hours. The potential side effects of infant exposure of these medications include myelosuppression, hypersensitivity reactions, nephrotoxicity, and neurotoxicity. It would be inadvisable for a mother to breastfeed while undergoing therapy with these 2 medications.

Self-assembly of thin plates from micrococcal nuclease-digested chromatin of metaphase chromosomes.

The three-dimensional organization of the enormously long DNA molecules packaged within metaphase chromosomes has been one of the most elusive problems in structural biology. Chromosomal DNA is associated with histones and different structural models consider that the resulting long chromatin fibers are folded forming loops or more irregular three-dimensional networks. Here, we report that fragments of chromatin fibers obtained from human metaphase chromosomes digested with micrococcal nuclease associate spontaneously forming multilaminar platelike structures. These self-assembled structures are identical to the thin plates found previously in partially denatured chromosomes. Under metaphase ionic conditions, the fragments that are initially folded forming the typical 30-nm chromatin fibers are untwisted and incorporated into growing plates. Large plates can be self-assembled from very short chromatin fragments, indicating that metaphase chromatin has a high tendency to generate plates even when there are many discontinuities in the DNA chain. Self-assembly at 37°C favors the formation of thick plates having many layers. All these results demonstrate conclusively that metaphase chromatin has the intrinsic capacity to self-organize as a multilayered planar structure. A chromosome structure consistent of many stacked layers of planar chromatin avoids random entanglement of DNA, and gives compactness and a high physical consistency to chromatids.

Irregular orientation of nucleosomes in the well-defined chromatin plates of metaphase chromosomes.

In previous studies with partially denatured metaphase chromosomes, we detected platelike structures instead of the chromatin fibers currently considered in different structural models for chromosomes. Here we have observed that dilution of compact metaphase chromosomes with hyposmotic solutions can transform whole chromatids into extended plates formed by many layers. Since this treatment is soft and it does not change the ionic conditions, these observations indicate that native chromosomes are formed by stacked plates. This strengthens our hypothesis about the multilayer structure of chromosomes, which was originally based on results obtained using stronger denaturing conditions. We have investigated the structure of plates emanated from chromosomes using electron tomography. Our three-dimensional reconstructions demonstrate conclusively that the surface of the plates is very smooth and do not show repetitive structures supporting any regular organization of nucleosomes; even the nucleosomes in plate edges show irregular orientations. Furthermore, we have used polarizing microscopy for the study of whole chromosomes in metaphase cells in aqueous solution. Our results show that condensed chromosomes are not birefringent under structuring ionic conditions similar to those used with plates. This observation is incompatible with the existence of parallel columns of nucleosomes within chromosomes. In summary, we have not detected any regular orientation of nucleosomes, but at the same time, our results indicate that the bulk of chromatin in native chromosomes is organized forming very well-defined plates, in which the nucleosomes of the successive layers are interdigitated. Presumably, this dense structure is required for safe transfer of DNA to daughter cells.