CRISPR-Cas9 Knockout Screens Identify DNA Damage Response Pathways and BTK as Essential for Cisplatin Response in Diffuse Large B-Cell Lymphoma.

The recurrence of diffuse large B-cell lymphoma (DLBCL) has been observed in 40% of cases. The standard of care for refractory/relapsed DLBCL (RR-DLBCL) is platinum-based treatment prior to autologous stem cell transplantation; however, the prognosis for RR-DLBCL patients remains poor. Thus, to identify genes affecting the cisplatin response in DLBCL, cisplatin-based whole-genome CRISPR-Cas9 knockout screens were performed in this study. We discovered DNA damage response (DDR) pathways as enriched among identified sensitizing CRISPR-mediated gene knockouts. In line, the knockout of the nucleotide excision repair genes XPA and ERCC6 sensitized DLBCL cells to platinum drugs irrespective of proliferation rate, thus documenting DDR as essential for cisplatin sensitivity in DLBCL. Functional analysis revealed that the loss of XPA and ERCC6 increased DNA damage levels and altered cell cycle distribution. Interestingly, we also identified BTK, which is involved in B-cell receptor signaling, to affect cisplatin response. The knockout of BTK increased cisplatin sensitivity in DLBCL cells, and combinatory drug screens revealed a synergistic effect of the BTK inhibitor, ibrutinib, with platinum drugs at low concentrations. Applying local and external DLBCL cohorts, we addressed the clinical relevance of the genes identified in the CRISPR screens. BTK was among the most frequently mutated genes with a frequency of 3-5%, and XPA and ERCC6 were also mutated, albeit at lower frequencies. Furthermore, 27-54% of diagnostic DLBCL samples had mutations in pathways that can sensitize cells to cisplatin. In conclusion, this study shows that XPA and ERCC6, in addition to BTK, are essential for the response to platinum-based drugs in DLBCL.

An idiosyncratic zonated stroma encapsulates desmoplastic liver metastases and originates from injured liver.

A perimetastatic capsule is a strong positive prognostic factor in liver metastases, but its origin remains unclear. Here, we systematically quantify the capsule's extent and cellular composition in 263 patients with colorectal cancer liver metastases to investigate its clinical significance and origin. We show that survival improves proportionally with increasing encapsulation and decreasing tumor-hepatocyte contact. Immunostaining reveals the gradual zonation of the capsule, transitioning from benign-like NGFRhigh stroma at the liver edge to FAPhigh stroma towards the tumor. Encapsulation correlates with decreased tumor viability and preoperative chemotherapy. In mice, chemotherapy and tumor cell ablation induce capsule formation. Our results suggest that encapsulation develops where tumor invasion into the liver plates stalls, representing a reparative process rather than tumor-induced desmoplasia. We propose a model of metastases growth, where the efficient tumor colonization of the liver parenchyma and a reparative liver injury reaction are opposing determinants of metastasis aggressiveness.

Small Cajal body-associated RNA 2 (scaRNA2) regulates DNA repair pathway choice by inhibiting DNA-PK.

Evidence that long non-coding RNAs (lncRNAs) participate in DNA repair is accumulating, however, whether they can control DNA repair pathway choice is unknown. Here we show that the small Cajal body-specific RNA 2 (scaRNA2) can promote HR by inhibiting DNA-dependent protein kinase (DNA-PK) and, thereby, NHEJ. By binding to the catalytic subunit of DNA-PK (DNA-PKcs), scaRNA2 weakens its interaction with the Ku70/80 subunits, as well as with the LINP1 lncRNA, thereby preventing catalytic activation of the enzyme. Inhibition of DNA-PK by scaRNA2 stimulates DNA end resection by the MRN/CtIP complex, activation of ATM at DNA lesions and subsequent repair by HR. ScaRNA2 is regulated in turn by WRAP53ß, which binds this RNA, sequestering it away from DNA-PKcs and allowing NHEJ to proceed. These findings reveal that RNA-dependent control of DNA-PK catalytic activity is involved in regulating whether the cell utilizes NHEJ or HR.

Neutralization of the Positive Charges on Histone Tails by RNA Promotes an Open Chromatin Structure.

RNA associates extensively with chromatin and can influence its structure; however, the potential role of the negative charges of RNA on chromatin structure remains unknown. Here, we demonstrate that RNA prevents precipitation of histones and can attenuate electrostatic interactions between histones and DNA, thereby loosening up the chromatin structure. This effect is independent of the sequence of RNA but dependent on its single-stranded nature, length, concentration, and negative charge. Opening and closure of chromatin by RNA occurs rapidly (within minutes) and passively (in permeabilized cells), in agreement with electrostatics. Accordingly, chromatin compaction following removal of RNA can be prevented by high ionic strength or neutralization of the positively charged histone tails by hyperacetylation. Finally, LINE1 repeat RNAs bind histone H2B and can decondense chromatin. We propose that RNA regulates chromatin opening and closure by neutralizing the positively charged tails of histones, reducing their electrostatic interactions with DNA.

Phosphorylation of the Cajal body protein WRAP53ß by ATM promotes its involvement in the DNA damage response.

The cellular response to DNA double-strand breaks is orchestrated by the protein kinase ATM, which phosphorylates key actors in the DNA repair network. WRAP53ß is a multifunctional protein that controls trafficking of factors to Cajal bodies, telomeres and DNA double-strand breaks but what regulates the involvement of WRAP53ß in these separate processes remains unclear. Here, we show that in response to various types of DNA damage, including IR and UV, WRAP53ß is phosphorylated on serine residue 64 by ATM with a time-course that parallels its accumulation at DNA lesions. Interestingly, recruitment of phosphorylated WRAP53ß (pWRAP53ßS64) to sites of such DNA damage promotes its interaction with γH2AX at these locations. Moreover, pWRAP53ßS64 stimulates the accumulation of the repair factor 53BP1 at DNA double-strand breaks and enhances repair of this type of damage via homologous recombination and non-homologous end joining. At the same time, phosphorylation of WRAP53ß is dispensable for its localization to Cajal bodies, where it accumulates even in unstressed cells. These findings not only reveal ATM to be an upstream regulator of WRAP53ß, but also indicates that phosphorylation of WRAP53ß at serine 64 controls its involvement in the DNA damage response and may also restrict its other functions.

Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3'-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing.

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5'-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5'-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression.

Surfactin restores and enhances swarming motility under heavy metal stress.

The present work reports the importance of lipopeptide biosurfactant on swarming motility of multi-metal resistant (MMR) bacterium under heavy metal stress. The MMR bacteria strain CM100B, identified as Bacillus cereus, was isolated from the coal mine sample. The strain was able to grow and reduce several metals namely Cd(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+) and Pb(2+) ions which are common environmental pollutants. Presence of toxic heavy metal ions in the swarming medium significantly altered the motility of CM100B. Presence of Cd(2+) and Pb(2+) ions inhibited development of peritrichous flagella, thus inhibiting swarming motility. However, the addition of anionic biosurfactant surfactin restored (in case of Cd(2+) and Pb(2+) ions) or enhanced (in case of Co(2+), Cu(2+), Ni(2+) and Mn(2+)) the swarming ability of CM100B. Zeta potential studies for determining bacterial cell surface charge indicated that surfactin provided a suitable swarming environment to bacteria even under metal stress by chelating to cationic metal ions. Non-ionic surfactant Triton X-100 was unable to restore swarming under Cd(2+) and Pb(2+) ion stress. Thus, suggesting that surfactin can aid in motility not only by reducing the surface tension of swarming medium but also by binding to metal ions in the presence of metal ions stress.

Eight nucleotide substitutions inhibit splicing to HPV-16 3'-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human keratinocytes.

The most commonly used 3'-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16.

Selenite stress elicits physiological adaptations in Bacillus sp. (strain JS-2).

A bacterial isolate (strain JS-2) characterized as Bacillus sp. was challenged with high concentrations of toxic selenite ions. The microbe was found to transform the toxic, soluble, colorless selenite (SeO(3)(2-)) oxyions to nontoxic, insoluble, red elemental selenium (Se(0)). This process of biotransformation was accompanied by cytoplasmic and surface accumulation of electron dense selenium (Se(0)) granules, as revealed in electron micrographs. The cells grown in the presence of selenite oxyions secreted large quantities of extracellular polymeric substances (EPS). There were quantitative and qualitative differences in the cell wall fatty acids of the culture grown in the presence of selenite ions. The relative percentage of total saturated fatty acid and cyclic fatty acid increased significantly, whereas the amount of total unsaturated fatty acids decreased when the cells were exposed to selenite stress. All these physiological adaptive responses evidently indicate a potentially important role of cell wall fatty acids and extracellular polymeric substances in determining bacterial adaptation towards selenite-induced toxicity, which thereby explains the remarkable competitiveness and ability of this microbe to survive the environmental stress.

Agrococcus carbonis sp. nov., isolated from soil of a coal mine.

An actinobacterial strain, designated G4(T), isolated from a coal mine was subjected to polyphasic taxonomic characterization. Cells were Gram-stain-positive, yellow-pigmented, non-motile and non-spore-forming cocci. This organism possessed a type B peptidoglycan with diaminobutyric acid as diagnostic diamino acid. The major respiratory quinones were MK-9, MK-10 and MK-11. The major fatty acids were anteiso-C(15 : 0) (41.6 %) and anteiso-C(17 : 0) (32.8 %). The predominant cellular polar lipids were diphosphatidylglycerol and phosphatidylglycerol. Cell wall sugars comprised galactose, glucose, ribose and rhamnose. 16S rRNA gene sequence analysis of strain G4(T) showed high similarity with Agrococcus baldri (98.9 %), Agrococcus citreus (97.8 %), Agrococcus jenensis (97.3 %) and Agrococcus terreus (97.0 %). Sequence similarity with the type strains of the other species of the genus Agrococcus was less than 97.0 %. The DNA-DNA relatedness of strain G4(T) with the type strains of Agrococcus baldri, Agrococcus citreus, Agrococcus jenensis and Agrococcus terreus was less than 70 %. On the basis of the physiological, biochemical and chemotaxonomic characteristics, strain G4(T) should be classified as the type strain of a novel species of the genus Agrococcus, for which the name Agrococcus carbonis sp. nov. is proposed. The type strain is G4(T) ( = MTCC 10213(T)  = DSM 22965(T)).

Yaniella fodinae sp. nov., isolated from a coal mine.

An orange bacterial strain, designated G5(T), was isolated during the study of the bacterial diversity of a coal mine. The cell wall of strain G5(T) contained peptidoglycan type A4α (l-Lys-Gly-l-Glu) and the sugars xylose and mannose. The major menaquinones were MK-8 (45.0 %) and MK-9 (34.0 %) and minor amounts of MK-7 and MK-8(H(2)) were also found. The major fatty acids were anteiso-C(15 : 0) (44.9 %) and iso-C(15 : 0) (44.2 %). The main cellular polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. 16S rRNA gene sequence analysis showed that strain G5(T) was closely related to Yaniella halotolerans YIM 70085(T) and Yaniella flava YIM 70178(T) (both 96.7 % 16S rRNA gene sequence similarity). The genomic DNA G+C content of strain G5(T) was 61.6 mol%. These data and other phenotypic characteristics clearly indicated that strain G5(T) represents a novel species of the genus Yaniella, for which the name Yaniella fodinae sp. nov. is proposed. The type strain is G5(T) (=MTCC 9846(T)=DSM 22966(T)).

Description of a novel actinobacterium Microbacterium assamensis sp. nov., isolated from water sample collected from the river Brahmaputra, Assam, India.

A Gram-positive, yellow pigmented actinobacterium, strain S2-48(T) was isolated from water sample collected from the river Brahmaputra, Assam, India and subjected to a polyphasic taxonomic study. Most of the physiological and biochemical properties, major fatty acids (C(15:0) Anteiso, iso C(16:0) and C(17:0) Anteiso), estimated DNA G+C content (70.2 mol%) and 16S rRNA gene sequence analysis showed that strain S2-48(T) belonged to the genus Microbacterium. Strain S2-48(T) exhibited highest 16S rRNA gene sequence similarity with Microbacterium testaceum (97.0%); however, the DNA-DNA relatedness value between strain S2-48(T) and M. testaceum was 9.1%. On the basis of differential phenotypic characteristics and genotypic distinctiveness, strain S2-48(T) should be classified within the genus Microbacterium as a novel species, for which the name Microbacterium assamensis is proposed. The type strain is S2-48(T) (=MTCC 10486(T) = DSM 23998(T)).

Aerobic biogenesis of selenium nanospheres by Bacillus cereus isolated from coalmine soil.

BACKGROUND: Microorganisms that are exposed to pollutants in the environment, such as metals/metalloids, have a remarkable ability to fight the metal stress by various mechanisms. These metal-microbe interactions have already found an important role in biotechnological applications. It is only recently that microorganisms have been explored as potential biofactories for synthesis of metal/metalloid nanoparticles. Biosynthesis of selenium (Se 0) nanospheres in aerobic conditions by a bacterial strain isolated from the coalmine soil is reported in the present study. RESULTS: The strain CM100B, identified as Bacillus cereus by morphological, biochemical and 16S rRNA gene sequencing [GenBank:GU551935.1] was studied for its ability to generate selenium nanoparticles (SNs) by transformation of toxic selenite (SeO3(2-)) anions into red elemental selenium (Se 0) under aerobic conditions. Also, the ability of the strain to tolerate high levels of toxic selenite ions was studied by challenging the microbe with different concentrations of sodium selenite (0.5 mM-10 mM). ESEM, AFM and SEM studies revealed the spherical Se 0 nanospheres adhering to bacterial biomass as well as present as free particles. The TEM microscopy showed the accumulation of spherical nanostructures as intracellular and extracellular deposits. The deposits were identified as element selenium by EDX analysis. This is also indicated by the red coloration of the culture broth that starts within 2-3 h of exposure to selenite oxyions. Selenium nanoparticles (SNs) were further characterized by UV-Visible spectroscopy, TEM and zeta potential measurement. The size of nanospheres was in the range of 150-200 nm with high negative charge of -46.86 mV. CONCLUSIONS: This bacterial isolate has the potential to be used as a bionanofactory for the synthesis of stable, nearly monodisperse Se 0 nanoparticles as well as for detoxification of the toxic selenite anions in the environment. A hypothetical mechanism for the biogenesis of selenium nanoparticles (SNs) involving membrane associated reductase enzyme(s) that reduces selenite (SeO3(2-)) to Se 0 through electron shuttle enzymatic metal reduction process has been proposed.