The CRISPR RNA-guided surveillance complex in Escherichia coli accommodates extended RNA spacers.

Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Finally, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.

Oxidation State of Capping Agent Affects Spatial Reactivity on Gold Nanorods.

Despite enormous progress toward controlling the shapes and surface chemistry of colloidal nanoparticles, spatial control of nanoparticle surface chemistry remains a major challenge. In recent years, there have been tantalizing reports demonstrating anisotropic silica coating of gold nanorods in which silica is deposited only on the sides by functionalizing the nanorods with poly(ethylene glycol) methyl ether thiol (PEG-thiol) prior to silica coating, but such results have been difficult to reproduce. We report that the oxidation state of PEG-thiol is key to anisotropic silica coating, with the disulfide, not the thiol, leading to side silica coating. PEG-disulfide appears to selectively functionalize the ends of gold nanorods, and robust methods are developed to reliably deposit side silica shells on PEG-disulfide functionalized gold nanorods.

Endometriosis and risk of ovarian and endometrial cancers in a large prospective cohort of U.S. nurses.

PURPOSE: Endometriosis is associated with ovarian cancer, but the relation with endometrial cancer is unclear. Prior studies generally were retrospective and had potential limitations, including use of self-reported endometriosis, failure to account for delays between symptom onset and endometriosis diagnosis, and changes in risk factors post-endometriosis diagnosis. We evaluated whether these limitations obscured a weak association with endometrial cancer and the extent to which these limitations impacted associations with ovarian cancer. METHODS: Cox proportional hazards regression models were used to assess associations between endometriosis and cancer risk, evaluating the impacts of self-reported vs. laparoscopically confirmed endometriosis, delayed diagnosis, and post-endometriosis diagnosis changes in risk factor exposures on relative risk estimates. RESULTS: Over 18 years of follow-up, we identified 228 ovarian and 166 endometrial cancers among 102,025 and 97,109 eligible women, respectively. Self-reported endometriosis was associated with ovarian cancer [relative risk (RR): 1.81; 95% confidence interval (CI): 1.26-2.58]; this association was stronger for laparoscopically confirmed endometriosis (HR: 2.14; 95% CI 1.45-3.15). No association was observed with endometrial cancer (self-report RR: 0.78; 95% CI 0.42-1.44; laparoscopic-confirmation RR: 0.76; 95% CI 0.35-1.64). Accounting for diagnosis delays or post-endometriosis diagnosis changes in risk factors had a little impact. CONCLUSIONS: This study adds to the evidence that endometriosis is not strongly linked to endometrial cancer risk and that the association with ovarian cancer is robust to misclassification, diagnostic delay, and changes in exposures post-endometriosis diagnosis. Our analysis suggests that confounding and misclassification do not obscure a weak association for endometrial cancer risk, although our results should be replicated.

Protein Adsorption to Charged Gold Nanospheres as a Function of Protein Deformability.

The corona that forms as protein adsorbs to gold nanospheres (AuNSs) is directly influenced by the surface chemistry of the AuNS. Tools to predict adsorption outcomes are needed for intelligent design of nanomaterials for biological applications. We hypothesized that the denaturation behavior of a protein might be a useful predictor of adsorption behavior to AuNSs, and used this idea to study protein adsorption to anionic citrate-capped AuNSs and to cationic poly(allylamine hydrochloride) (PAH) wrapped AuNSs. Three proteins (α-amylase (A-Amy), ß-lactoglobulin (BLG), and bovine serum albumin (BSA)), representing three different classes of acid denaturation behavior, were selected with BLG being the least deformable and BSA being the most deformable. Protein adsorption to AuNSs was monitored via UV-vis spectrophotometry and dynamic light scattering. Changes to the protein structure upon AuNS interaction were monitored via circular dichroism spectroscopy. Binding constants were determined using the Langmuir adsorption isotherm, resulting in BSA > BLG ≫ A-Amy affinities for citrate-capped gold nanospheres. PAH-coated AuNSs displayed little affinity for these proteins at similar concentrations as citrate-coated AuNSs and became agglomerated at high protein concentrations. The enzymatic activity of A-Amy/citrate AuNS conjugates was measured via colorimetric assay, and found to be 11% of free A-Amy, suggesting that binding restricts access to the active site. Across both citrate AuNSs and PAH AuNSs, the changes in secondary structure were greatest for BSA > A-Amy > BLG, which does follow the trends predicted by acid denaturation characteristics.

Surface Chemistry of Gold Nanorods.

Gold nanorods have garnered a great deal of scientific interest because of their unique optical properties, and they have the potential to greatly impact many areas of science and technology. Understanding the structure and chemical makeup of their surfaces as well as how to tailor them is of paramount importance in the development of their successful applications. This Feature Article reviews the current understanding of the surface chemistry of as-synthesized gold nanorods, methods of tailoring the surface chemistry of gold nanorods with various inorganic and organic coatings/ligands, and the techniques employed to characterize ligands on the surface of gold nanorods as well as the associated measurement challenges. Specifically, we address the challenges of determining how thick the ligand shell is, how many ligands per nanorod are present on the surface, and where the ligands are located in regiospecific and mixed-ligand systems. We conclude with an outlook on the development of the surface chemistry of gold nanorods leading to the development of a synthetic nanoparticle surface chemistry toolbox analogous to that of synthetic organic chemistry and natural product synthesis.

Comparison of age at natural menopause in BRCA1/2 mutation carriers with a non-clinic-based sample of women in northern California.

BACKGROUND: Germline mutations in BRCA1 and BRCA2 (BRCA1/2) are related to an increased lifetime risk of developing breast and ovarian cancer. Although risk-reducing salpingo-oophorectomy reduces the risk of both cancers, loss of fertility is a major concern. A recent study suggested an association between BRCA1 mutation and occult primary ovarian insufficiency. The objective of the current study was to determine whether BRCA1/2 mutation carriers have an earlier onset of natural menopause compared with unaffected women. METHODS: White carriers of the BRCA1/2 gene (n = 382) were identified within the Breast Cancer Risk Program Registry at the University of California at San Francisco and compared with non-clinic-based white women in northern California (n = 765). The 2 groups were compared with regard to median age at the time of natural menopause before and after adjustment for known risk factors, and the role of smoking within each group was examined using the Kaplan-Meier approach for unadjusted analyses and Cox proportional hazards regression analyses for adjusted analyses. RESULTS: The median age at the time of natural menopause in the BRCA1/2 carriers was significantly younger than among the unaffected sample (50 years vs 53 years; P < .001). The unadjusted hazard ratio for natural menopause when comparing BRCA1/2 carriers with unaffected women was 4.06 (95% confidence interval, 3.03-5.45) and was 3.98 (95% confidence interval, 2.87-5.53) after adjusting for smoking, parity, and oral contraceptive use. For BRCA1/2 carriers who were current heavy smokers (smoking ≥ 20 cigarettes/day), the median age at natural menopause was 46 years versus 49 years for nonsmokers (P = .027). CONCLUSIONS: The BRCA1/2 mutation was associated with a significantly earlier age at natural menopause, and heavy smoking compounded this risk. Because the relationship between menopause and the end of natural fertility is considered to be fixed, these findings suggest the risk of earlier infertility among BRCA1/2 carriers.

Modeling Liver Organogenesis by Recreating Three-Dimensional Collective Cell Migration: A Role for TGFß Pathway.

Three-dimensional (3D) collective cell migration (CCM) is critical for improving liver cell therapies, eliciting mechanisms of liver disease, and modeling human liver development and organogenesis. Mechanisms of CCM differ in 2D vs. 3D systems, and existing models are limited to 2D or transwell-based systems, suggesting there is a need for improved 3D models of CCM. To recreate liver 3D CCM, we engineered in vitro 3D models based upon a morphogenetic transition that occurs during liver organogenesis, which occurs rapidly between E8.5 and E9.5 (mouse). During this morphogenetic transition, 3D CCM exhibits co-migration (multiple cell types), thick-strand interactions with surrounding septum transversum mesenchyme (STM), branching morphogenesis, and 3D interstitial migration. Here, we engineer several 3D in vitro culture systems, each of which mimics one of these processes in vitro. In mixed spheroids bearing both liver cells and uniquely MRC-5 (fetal lung) fibroblasts, we observed evidence of co-migration, and a significant increase in length and number of liver spheroid protrusions, which was highly sensitive to transforming growth factor beta 1 (TGFß1) stimulation. In MRC-5-conditioned medium (M-CM) experiments, we observed dose-dependent branching morphogenesis associated with an upregulation of Twist1, which was inhibited by a broad TGFß inhibitor. In models in which liver spheroids and MRC-5 spheroids were co-cultured, we observed complex strand morphogenesis, whereby thin, linear, 3D liver cell strands attach to the MRC-5 spheroid, anchor and thicken to form permanent and thick anchoring contacts between the two spheroids. In these spheroid co-culture models, we also observed spheroid fusion and strong evidence for interstitial migration. In conclusion, we present several novel cultivation systems that recreate distinct features of liver 3D CCM. These methodologies will greatly improve our molecular, cellular, and tissue-scale understanding of liver organogenesis, liver diseases like cancer, and liver cell therapy, and will also serve as a tool to bridge conventional 2D studies and preclinical in vivo studies.

Large scale self-assembly of plasmonic nanoparticles on deformed graphene templates.

Hierarchical heterostructures of two-dimensional (2D) nanomaterials are versatile platforms for nanoscale optoelectronics. Further coupling of these 2D materials with plasmonic nanostructures, especially in non-close-packed morphologies, imparts new metastructural properties such as increased photosensitivity as well as spectral selectivity and range. However, the integration of plasmonic nanoparticles with 2D materials has largely been limited to lithographic patterning and/or undefined deposition of metallic structures. Here we show that colloidally synthesized zero-dimensional (0D) gold nanoparticles of various sizes can be deterministically self-assembled in highly-ordered, anisotropic, non-close-packed, multi-scale morphologies with templates designed from instability-driven, deformed 2D nanomaterials. The anisotropic plasmonic coupling of the particle arrays exhibits emergent polarization-dependent absorbance in the visible to near-IR regions. Additionally, controllable metasurface arrays of nanoparticles by functionalization with varying polymer brushes modulate the plasmonic coupling between polarization dependent and independent assemblies. This self-assembly method shows potential for bottom-up nanomanufacturing of diverse optoelectronic components and can potentially be adapted to a wide array of nanoscale 0D, 1D, and 2D materials.

Cooperate or Not Cooperate in Predictable but Periodically Varying Situations? Cooperation in Fast Oscillating Environment.

In this work, the cooperation problem between two populations in a periodically varying environment is discussed. In particular, the two-population prisoner's dilemma game with periodically oscillating payoffs is discussed, such that the time-average of these oscillations over the period of environmental variations vanishes. The possible overlaps of these oscillations generate completely new dynamical effects that drastically change the phase space structure of the two-population evolutionary dynamics. Due to these effects, the emergence of some level of cooperators in both populations is possible under certain conditions on the environmental variations. In the domain of stable coexistence the dynamics of cooperators in each population form stable cycles. Thus, the cooperators in each population promote the existence of cooperators in the other population. However, the survival of cooperators in both populations is not guaranteed by a large initial fraction of them.

Interaction of Alpha-Synuclein and Its Mutants with Rigid Lipid Vesicle Mimics of Varying Surface Curvature.

Abnormal aggregation of alpha-synuclein (α-syn), an intrinsically disordered neuronal protein, is strongly implicated in the development of Parkinson's disease. Efforts to better understand α-syn's native function and its pathogenic role in neurodegeneration have revealed that the protein interacts with anionic lipid vesicles via adoption of an amphipathic α-helical structure; however, the ability of α-syn to remodel lipid membranes has made it difficult to decipher the role of vesicle surface curvature in protein binding behavior. In this study, sodium dodecyl sulfate (SDS)-coated gold nanoparticles (AuNPs), which mimic bilayer vesicle architecture, were synthesized in order to conduct a systematic investigation into the binding interaction of α-syn and two of its mutants (A30P and E46K) with rigid lipid vesicle mimics of defined surface curvature. By incorporating a rigid AuNP core (∼10-100 nm), the ability of α-syn to remodel the vesicle mimics was removed and their surface curvature could be fixed. Proteomics studies showed that, upon binding of free α-syn to the surface of SDS-AuNPs, the N-terminus of α-syn became less solvent accessible, whereas its C-terminus became more accessible. Interestingly, α-syn's non-amyloid-ß component (NAC) region also exhibited increased solvent accessibility, suggesting that α-syn bound to rigid vesicle-like structures could possess heightened aggregation propensity and therefore pathogenicity. Additionally, both the A30P and E46K mutations were found to adopt distinct binding modes on the mimics' surface. In contrast with previous reports, similar binding affinities were observed for WT, A30P, and E46K α-syn toward SDS-AuNPs of all sizes, indicating the potential importance of vesicle deformability in determining α-syn binding behavior.

A Demonstration of Le Chatelier's Principle on the Nanoscale.

Photothermal desorption of molecules from plasmonic nanoparticles is an example of a light-triggered molecular release due to heating of the system. However, this phenomenon ought to work only if the molecule-nanoparticle interaction is exothermic in nature. In this study, we compare protein adsorption behavior onto gold nanoparticles for both endothermic and exothermic complexation reactions, and demonstrate that Le Chatelier's principle can be applied to predict protein adsorption or desorption on nanomaterial surfaces. Polyelectrolyte-wrapped gold nanorods were used as adsorption platforms for two different proteins, which we were able to adsorb/desorb from the nanorod surface depending on the thermodynamics of their interactions. Furthermore, we show that the behaviors hold up under more complex biological environments such as fetal bovine serum.

Ovarian Aging in Women With BRCA Germline Mutations.

Context: Recent clinical and laboratory studies suggested that women with BRCA mutations have lower ovarian reserve and their primordial follicle oocytes may be more prone to DNA damage; however, direct proof is lacking. Objective: To determine whether women with germline BRCA mutations have reduced primordial follicle reserve and increased oocyte DNA damage. Design: A comparative laboratory study of ovarian tissue obtained from unaffected BRCA mutation carriers (BMCs) vs age-matched organ donor cadavers. Setting: Two academic centers. Patients or Other Participants: Of the 230 ovarian specimens from BMCs, 18 met the study inclusion criteria. Healthy ovaries from 12 organ donor cadavers served as controls. Intervention: Histology and immunohistochemical analysis on paraffin-embedded ovarian sections. Main Outcome Measure(s): Primordial follicle density and the percentage of DNA double-strand break (DSB)-positive primordial follicle oocytes. Results: Ovaries from BMCs had significantly lower primordial follicle densities than those of controls (11.2 ± 2.0 vs 44.2 ± 6.2 follicles/mm3; P = 0.0002). BRCA mutations were associated with increased DNA DSBs in primordial follicle oocytes (62% ± 5.2% vs 36% ± 3.4%; P = 0.0005). In subgroup analyses, both BRCA1 and BRCA2 mutations were associated with lower primordial follicle density (P = 0.0001 and 0.0030, respectively), and BRCA1 mutations were associated with higher DNA DSBs (P = 0.0003) than controls. The rates of follicle decline (R2 = 0.74; P = 0.0001) and DNA DSB accumulation (R2 = 0.70; P = 0.0001) appeared to be accelerated, particularly in primordial follicle oocytes of BMCs over age 30 years. Conclusions: We provide direct evidence of diminished ovarian reserve as well as accelerated primordial follicle loss and oocyte DNA damage in women with BRCA mutations. These findings may further our understanding of ovarian aging, and be useful when counseling BMCs.

Anisotropic Nanoparticles and Anisotropic Surface Chemistry.

Anisotropic nanoparticles are powerful building blocks for materials engineering. Unusual properties emerge with added anisotropy-often to an extraordinary degree-enabling countless new applications. For bottom-up assembly, anisotropy is crucial for programmability; isotropic particles lack directional interactions and can self-assemble only by basic packing rules. Anisotropic particles have long fascinated scientists, and their properties and assembly behavior have been the subjects of many theoretical studies over the years. However, only recently has experiment caught up with theory. We have begun to witness tremendous diversity in the synthesis of nanoparticles with controlled anisotropy. In this Perspective, we highlight the synthetic achievements that have galvanized the field, presenting a comprehensive discussion of the mechanisms and products of both seed-mediated and alternative growth methods. We also address recent breakthroughs and challenges in regiospecific functionalization, which is the next frontier in exploiting nanoparticle anisotropy.

Control of protein orientation on gold nanoparticles.

Gold nanoparticles (Au NPs) have attracted much attention due to their potential applications in nano-medicine. While numerous studies have quantified biomolecular adsorption to Au NPs in terms of equilibrium binding constants, far less is known about biomolecular orientation on nanoparticle surfaces. In this study, the binding of the protein α-synuclein to citrate and (16-mercaptohexadecyl) trimethylammonium bromide (MTAB) coated 12 nm Au NPs is examined by heteronuclear single quantum coherence NMR spectroscopy to provide site-specific measurements of protein-nanoparticle binding. Molecular dynamics simulations support the orientation assignments, which show N-terminus binding to the Au NP for citrate-capped NPs, and C-terminus binding for the MTAB-capped NPs.

α-Synuclein's adsorption, conformation, and orientation on cationic gold nanoparticle surfaces seeds global conformation change.

α-Synuclein (α-syn), an aggregation-prone amyloid protein, has been suggested as a potential cause of Parkinson's disease. When misfolded, α-syn aggregates as Lewy bodies in the brain, the loss of which can disrupt protein homeostasis. To investigate the potential of nanoparticle-mediated therapy for amyloid diseases, α-syn adsorption onto positively charged poly(allylamine hydrochloride) coated gold nanoparticles (PAH Au NPs) was studied. α-Syn adsorbs in multilayers onto PAH Au NPs, which with increasing α-syn/PAH Au NP ratios (>2000 α-syn/PAH Au NP) results in the flocculation and sedimentation of α-syn coated PAH Au NPs. The orientation and conformation of α-syn on PAH Au NPs were studied using trypsin digestion and circular dichroism, which showed that α-syn adopts a random orientation on PAH Au NPs, with an increase in ß-sheet and a decrease in α-helix structures. A consistent global change in α-syn's conformation was also observed regardless of PAH Au NP concentration, suggesting bound α-syn initiates conformational changes to free α-syn.