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Scrub typhus, is a mite-borne disease caused by bacteria named Orientia tsutsugamushi. In recent years the incidence of scrub typhus is increasing day by day. The disease is easily missed because of low sensitization among clinicians and non-specific clinical manifestation. The disease can be fatal in untreated cases. With the availability of testing methods, it can be easily diagnosed and treated. This study aims to depict the epidemiology of scrub typhus in southern Odisha.A total of 170 blood samples were collected from clinically suspected acute undifferentiated febrile illness (AUFI) cases. Samples were tested serologically for antibodies against Orientia tsutsugamushi by commercially available Immunochromatography test (ICT) and enzyme-linked immunosorbent assay (ELISA) kit as per the manufacturer's instruction. Molecular diagnosis was done by nested polymerase chain reaction (N-PCR) and Sanger sequencing was done to know the circulating strains.Out of 170 cases of AUFI, 74 cases were diagnosed scrub typhus by IgM ELISA and 67 were positive by ICT. Males were affected more and cases were more clustered in the Ganjam district. The disease followed a typical seasonal i.e. more cases were seen in cooler months of the year. Sequencing revealed the strains were Gilliam and Karp like.The burden of scrub typhus was 43.5% among the study population. Determining the serotypes in endemic areas is important for basic research on the classification of Orientia tsutsugamushi, the development of vaccines, and the definitive diagnosis of scrub typhus. Expanding the panel of antigens used to test scrub typhus and to take into account of local antigenic diversity would improve the sensitivity of serological diagnosis.

While the past decade has seen meaningful improvements in clinical outcomes for multiple myeloma patients, a subset of patients do not benefit from current therapeutics for unclear reasons. Many gene expression-based models of risk have been developed, but each model uses a different combination of genes and often involve assaying many genes making them difficult to implement. We organized the Multiple Myeloma DREAM Challenge, a crowdsourced effort to develop models of rapid progression in newly diagnosed myeloma patients and to benchmark these against previously published models. This effort lead to more robust predictors and found that incorporating specific demographic and clinical features improved gene expression-based models of high risk. Furthermore, post challenge analysis identified a novel expression-based risk marker and histone modifier,PHF19, which featured prominently in several independent models. Lastly, we show that a simple four feature predictor composed of age, International Staging System stage (ISS), and expression ofPHF19andMMSETperforms similarly to more complex models with many more gene expression features included.Key pointsMost comprehensive and unbiased assessment of prognostic biomarkers in MM resulting in a robust and parsimonious model.Identification ofPHF19as the expression based biomarker most strongly associated with rapid progression in MM patients.

INTRODUCTION Multiple myeloma (MM) remains an incurable disease with a median overall survival of approximately 5 years. Gain or amplification of 1q21 (1q21+) occurs in around 40% of patients with MM and generally portends a poor prognosis. Patients with MM who harbor 1q21+ are at increased risk of drug resistance, disease progression, and death. New pharmacotherapies with novel modes of action are required to overcome the negative prognostic impact of 1q21+. Areas covered: This review discusses the detection, biology, prognosis, and therapeutic targeting of 1q21+ in newly diagnosed and relapsed MM. Patients with MM and 1q21+ tend to present with higher tumor burden, greater end-organ damage, and more co-occurring high-risk cytogenetic abnormalities than patients without 1q21+. The chromosomal rearrangements associated with 1q21+ result in dysregulation of genes involved in oncogenesis. Identification and characterization of the 1q21+ molecular targets are needed to inform on prognosis and treatment strategy. Clinical trial data are emerging that addition of isatuximab to combination therapies may improve outcomes in patients with 1q21+ MM. Expert opinion: In the next 5 years, the results of ongoing research and trials are likely to focus on the therapeutic impact and treatment decisions associated with 1q21+ in MM.

A TPP1 mutation known to cause telomere shortening and bone marrow failure in humans recapitulates telomere loss but results in severe germline defects in mice without impacting murine hematopoiesis., Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita, frequently resulting in spontaneous bone marrow failure. A dyskeratosis congenita mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic and germline compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to infertility at steady-state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice.

Aim: The study was to evaluate the phytogenic chemical compounds and Antifungal Activity of essential oil from roots of Selinum vaginatum C.B. Clarke, growing in the Himalayan region of Jammu & Kashmir. Methodology: The essential oil was analyzed by Gas Chromatography & Gas Chromatography-Mass Spectrometry in relation with their Kavot indices and mass spectra. Results: The oil was found completely dominated by oxygenated sesquiterpenoids (71.9%) which includes 14-hydroxy-δ-cadinene (37.5%), khusinol (20.7%), viridiflorol (8.0%), acorenone -B (4.2%) and 14-oxy-α-muurolene (1.1%) whereas δ-cadinene (8.9%), α-copaene (6.8%), germacrene-A (2.5%), and β-caryophyllene (1.3%) were the major compounds among sesquiterpenoids. Monoterpenoids constituted as the minor portion (3.8%) of essential oil. The oil was found almost free from oxygenated monoterpenoids (0.2%). The roots of S. vaginatum are used in folk lore medicines in Jammu & Kashmir. The oil from the roots showed marked antifungal activity. The oil had shown 100% mycelia growth inhibition against A. tenuis, C. graminicola, R. solani and S. sclerotiorum at a concentration of 500 µg/ml, 2000 µg/mL, 2000 µg/mL and 300 µg/mL respectively. However F. oxysporum was found less susceptible to the root oil of S. vaginatum. The IC50 values showed a range from 57.4 µg/mL–74.7 µg/mL as compared to standard fungicides with IC50 values 32.8 µg/mL–98.6 µg/mL. The spore germination inhibition test revealed the root oil as a potent inhibitor with IC50 values as 201.4 µg/mL, 414.7 µg/mL and 784.7 µg/mL for A. tenuis, C. graminicola and F. oxysporum. Conclusion: Our study showed that14-hydroxy-δ-cadinene (37.5%), khusinol (20.7%), & viridiflorol (8.0%) are the major components in this oil and possessed potent antifungal activity against test fungal strain, respectively.

Formation of individualized sister chromatids is essential for their accurate segregation. In budding yeast, while most of the genome segregates at the metaphase to anaphase transition, resolution of the ribosomal DNA (rDNA) repeats is delayed. The timing and mechanism in human cells is unknown. Here we show that resolution of human rDNA occurs in anaphase after the bulk of the genome, dependent on tankyrase 1, condensin II, and topoisomerase IIα. Defective resolution leads to rDNA bridges, rDNA damage, and aneuploidy of an rDNA-containing acrocentric chromosome. Thus, temporal regulation of rDNA segregation is conserved between yeast and man and is essential for genome integrity.

Non-stochastic hesitation in fuzzy time series forecasting methods occurs due to availability of more than one fuzzification methods of time series data. Recently hesitant fuzzy set has gained attention of the researchers to address issue of aforesaid non-stochastic hesitation. In this research paper, we propose and develop a computational algorithm-based method for financial time series forecasting using hesitant fuzzy set. The proposed method uses hesitant fuzzy logical relations that are constructed using triangular fuzzy sets with equal and unequal intervals. In the proposed forecasting method, hesitant fuzzy logical relations are aggregated using a hesitant fuzzy aggregation operator. Advantages of developed hesitant fuzzy set based forecasting method are that it is easy to implement, can cope with huge time series database and enhances the accuracy in financial time series forecast. To see the performance of proposed method in financial time series forecasting, it is implemented on two financial experimental dataset of TAIFEX and SBI share price at BSE. Rigorous comparison analysis of the proposed forecasting method is done by comparing it with other conventional and computational fuzzy time series forecasting methods in terms of RMSE, AFER. Significance of accuracy enhancement in forecasted outputs is also verified using two-tailed t test.

In this chapter, the authors study a weighted-((f / (r, s)), k)/ (m, n): G system. The system consists of mn components arranged in a matrix form and the system works if all the sub matrices of order (r, s), the total weight of the working components is greater than f and the total weight of the working components in the system is at least k. This chapter deals with the evaluation of fuzzy reliability and fuzzy mean time to failure of the considered system with the application of fuzzy universal generating function and fuzzy Rayleigh distribution. In this study, the authors formed some prepositions to understand the behaviour of the considered system with respect to different varying parameters and also present an illustrative example to understand them.

The establishment of heterochromatin in fission yeast involves methyltransferase Clr4-mediated H3-Lys9 methylation, which is bound specifically by Swi6/HP1. However, the mechanism of propagation of heterochromatin through multiple cell divisions is not known. A role of DNA replication in propagating the heterochromatin is envisaged. Studies inS. pombehave indicated a direct interaction between DNA Polα and Swi6/HP1 and between DNA Polε and Rik1-Dos2 complex, suggesting a coupling between DNA replication and heterochromatin assembly. Here, we show that like DNA Polα, Polδ, which plays a role in both leading and lagging strand replication, also plays a role in silencing at mating type and centromere. We show that both the polymerases α and δ interact directly with both Clr4 and Swi6/HP1. Mutations in both the polymerases lead to decrease in H3-Lys9 methylation and Swi6 at the mating type and left outer repeats of centromeres I and II, with a reciprocal increase in their level at the central element,cnt, at all the three centromeres. These mutations also cause defects in chromosome segregation, recruitment of Cohesin and chromosome dynamics during mitosis and meiosis. Thus, our results indicate that a tight coordination between DNA replication machinery and propagation of the heterochromatin-specific epigenetic mark.

Rickettsial diseases (RDs) are major under-diagnosed causes of arthropod borne acute febrile illness (AFI) presenting with a range of symptoms from mild self-limiting fever to fatal sepsis. The spotted fever group (SFG) and typhus group (TG) are major RDs, which are commonly caused by Rickettsia conorii and Rickettsia typhi, respectively. The limited availability and role of serological tests in the acute phase of illness warrants rapid reliable molecular methods for diagnosis and epidemiological studies. Two hundred patients with AFI in whom the routine fever diagnostics were negative, were enrolled over a period of two months (April 2019 to May 2019). DNA was extracted and in-house nested PCR using primers specific for both SPG and TG pathogens was used. The positive amplified products were sequenced for species identification and phylogenetic analysis was performed using MEGA 7.0.14 software (iGEM, Temple University, Philadelphia, PA 19122, USA). The demographic details of the RD cases were documented. The prevalence of RD among AFI cases was 7% (14/200); SFG and TG were identified as the cause in 4% and 3% of AFI cases, respectively. The median age of the RD cases was 22 years (range 2–65). The median duration of fever was 3 days (range 1–12). The RD cases presented with respiratory symptoms or signs (44.44%), jaundice (22.22%), abdominal pain (22.22%), diarrhea (22.22), vesicular rash (11.11%), vomiting (11.11%), loss of appetite (11.11%), headache (11.11%), leukocytosis (88.88% with mean count 22,750/mm3), and thrombocytopenia (33.33%). The cases were treated empirically with piperacillin-tazobactam (66.66%), clindamycin (44.44%), cefotaxime (33.33%), meropenem (33.33%), metronidazole (33.33%), doxycycline (22.22%), azithromycin (22.22%), ceftriaxone (11.11%), and amoxicillin-clavulanic acid (11.11%). The mortality among the RD cases was 11.11%. The present pilot study shows that RD is not an uncommon cause of AFI in north India. The febrile episodes are usually transient, not severe and associated with heterogenous clinical presentation without documented history of tick exposure in the hospitalized patients. The transient, non-severe, febrile illness could be due to transient rickettsemia resulting from empirical antimicrobial therapy as the rickettsial organisms are expected to be more susceptible to higher doses of β-lactam antibiotics. The study emphasizes the molecular method as a useful tool to identify rickettsial etiology in AFI.

Intuitionistic fuzzy sets (IFSs) have been proved to be more ideal than fuzzy sets to handle non-probabilistic uncertainty and non-determinism in the system. The present study proposes an IFS-based...

We propose a fuzzy time series forecasting method using hesitant fuzzy sets.An aggregation operator is also proposed to aggregate hesitant fuzzy elements.Hesitancy is introduced by using multiple fuzzification methods.University of Alabama enrollments and SBI share prices are forecasted.Performance of method is measured in terms of MSE and AFER. This study proposes a fuzzy time series forecasting method based on hesitant fuzzy sets for forecasting in the environment of hesitant information. The proposed method addresses the problem of establishing a common membership grade for the situation when multiple fuzzification methods are available to fuzzify time series data. An aggregation operator for aggregating hesitant information is also proposed in the study. The proposed method is implemented to forecast enrollment at University of Alabama and price of state bank of India (SBI) share at Bombay stock exchange (BSE), India. In both time series data are fuzzified with triangular fuzzy sets constructed using intervals of equal and unequal length. The performance of the proposed method in forecasting student enrollments and SBI share price is measured in terms of root mean square and average forecasting errors. Statistical validation and performance analysis is also carried out to validate the proposed forecasting method.

SummaryTelomerase replicates chromosome ends in germ and somatic stem cells to facilitate continued proliferation. Telomerase action depends on the telomeric protein TPP1, which recruits telomerase to telomeres and facilitates processive DNA synthesis. Here we identify separation-of-function long (TPP1-L) and short (TPP1-S) isoforms of TPP1 differing only in 86 amino acids at their N-terminus. While both isoforms retain the ability to recruit telomerase, only TPP1-S facilitates telomere synthesis. We identify a novel intragenic noncoding RNA in the 3’-UTR of the TPP1-encoding gene that specifically shuts down telomerase activation-incompatible TPP1-L to establish TPP1-S as the predominant isoform in somatic cells. Strikingly, TPP1-L is the major isoform in testes, where it can function to restrain telomerase in mature germ cells. Our studies uncover how differential expression of two isoforms allows TPP1 to perform separate functions in different cells, and demonstrate how isoform choice can be determined by an intragenic noncoding RNA.

International audience; The developmental asymmetry of fission yeast daughter cells derives from inheriting older Watson' versus older Crick' DNA strand from the parental cell, strands that are complementary but not identical with each other. A novel DNA strand-specific imprint', installed during DNA replication at the mating-type locus (mat1), imparts competence for cell type inter-conversion to one of the two chromosome replicas. The catalytic subunit of DNA Polymerase (Pol) has been implicated in the imprinting process. Based on its known biochemical function, Pol might install the mat1 imprint during lagging strand synthesis. The nature of the imprint is not clear it is either a nick or a ribonucleotide insertion. Our investigations do not support a direct role of Pol in nicking through putative endonuclease domains but confirm its indirect role in installing an alkali-labile moiety as the imprint. While ruling out the role of the primase subunit of Pol holoenzyme, we find that mutations in the Pol-recruitment and putative primase homology domain in Mcm10/Cdc23 abrogate the ribonucleotide imprint formation. These results, while confirming the ribonucleotide nature of the imprint suggest the possibility of a direct role of Mcm10/Cdc23 in installing it in cooperation with Pol and Swi1.

In this chapter, an application of dual hesitant fuzzy set (DHFS) in intuitionistic fuzzy time series forecasting is proposed to handle fuzziness and non-determinism that occurs due to multiple valid fuzzification method for time series data. Advantages of the proposed DHFS-based time series forecasting method are that it includes characteristics of both intuitionistic and hesitant fuzzy sets to handle the non-determinism and hesitancy corresponding to single membership grade multiple membership grades of an element. In the present study, universe of discourse is partitioned and fuzzified the time series data by two different fuzzification methods (triangular and Gaussian) to construct DHFS. Further, elements of DHFS are aggregated to construct the intuitionistic fuzzy sets. Proposed method is implemented over the share market prizes of SBI at BSE, India and SENSEX of BSE to confirm its out performance over existing time series forecasting methods using RMSE and AFER.

Mammalian chromosomes terminate in stretches of repetitive telomeric DNA that act as buffers to avoid loss of essential genetic information during end-replication. A multiprotein complex known as shelterin prevents recognition of telomeric sequences as sites of DNA damage. Telomere erosion contributes to human diseases ranging from BM failure to premature aging syndromes and cancer. The role of shelterin telomere protection is less understood. Mutations in genes encoding the shelterin proteins TRF1-interacting nuclear factor 2 (TIN2) and adrenocortical dysplasia homolog (ACD) were identified in dyskeratosis congenita, a syndrome characterized by somatic stem cell dysfunction in multiple organs leading to BM failure and other pleiotropic manifestations. Here, we introduce the biochemical features and in vivo effects of individual shelterin proteins, discuss shelterin functions in hematopoiesis, and review emerging knowledge implicating the shelterin complex in hematological disorders.

Multiple myeloma (MM) is the second most common hematological cancer and is characterized by genetic features including translocations, chromosomal copy number aberrations, and mutations in key oncogene and tumor suppressor genes. Dysregulation of the tumor suppressor TP53 is important in the pathogenesis of many cancers, including MM. In newly-diagnosed MM patients, TP53 dysregulation occurs in three subsets: monoallelic deletion as part of deletion of chromosome 17p (del17p) (~8%), monoallelic mutations (~6%), and biallelic inactivation (~4%). Del17p is an established high-risk feature in MM and is included in current disease staging criteria. Biallelic inactivation and mutation have also been reported in MM patients but are not yet included in disease staging criteria for high-risk disease. Emerging clinical and genomics data suggest that the biology of high-risk disease is complex, and so far, traditional drug development efforts to target dysregulated TP53 have not been successful. Here we review the TP53 dysregulation literature in cancer and in MM, including the three segments of TP53 dysregulation observed in MM patients. We propose a reverse translational approach to identify novel targets and disease drivers from TP53 dysregulated patients to address the unmet medical need in this setting.

Summary: Telomerase replicates chromosome ends in germ and somatic stem cells to facilitate their continued proliferation. Telomerase action depends on the telomeric protein TPP1, which recruits telomerase to telomeres and facilitates processive DNA synthesis. Here, we identify separation-of-function long (TPP1-L) and short (TPP1-S) isoforms of TPP1 that appear to be generated from separate transcripts and differ only in 86 amino acids at their N terminus. Although both isoforms retain the ability to recruit telomerase, only TPP1-S facilitates efficient telomere synthesis. We find that TPP1-S is the predominant isoform in somatic cells, and strikingly, TPP1-L is the major isoform in differentiated male germ cells. We observed that TERT expression persists in these germ cells, suggesting that TPP1-L could restrain telomerase in this context. We show how differential expression of TPP1 isoforms determines telomerase function and demonstrate how alternative transcription start sites allow one gene to perform distinct functions in different biological contexts. : Human TPP1 is critical for telomerase function. Grill et al. demonstrate that TPP1 exists as two isoforms: TPP1-S and TPP1-L. TPP1-S, but not TPP1-L, activates telomerase. TPP1-S is the major isoform in all somatic cells, and TPP1-L is upregulated in differentiated germ cells to presumably curb telomerase that persists there. Keywords: telomerase, TPP1, somatic cell, stem cell, germ cell, cancer

The developmental asymmetry of fission yeast daughter cells derives from inheriting “older Watson” versus “older Crick” DNA strand from the parental cell, strands that are complementary but not identical with each other. A novel DNA strand-specific “imprint”, installed during DNA replication at the mating-type locus (mat1), imparts competence for cell type inter-conversion to one of the two chromosome replicas. The biochemical nature of the imprint and the mechanism of its installation are still not understood. The catalytic subunit of DNA Polymerase α (Polα) has been implicated in the imprinting process. Based on its known biochemical function, Polα might install themat1imprint during lagging strand synthesis. The nature of the imprint is not clear: it is either a nick or a ribonucleotide insertion. Our investigations do not support a role of Polα in nicking through putative endonuclease domains but confirm its role in installing an alkali-labile moiety as the imprint. A detailed genetic and molecular analysis reveals a direct role of the Cdc23/Mcm10 primase activity in installing the imprint in cooperation with Polα and Swi1.

An interval-valued intuitionistic uncertain linguistic set (IVIULS) combines the ambiguity, fuzziness as well as indeterminacy in real-life predicaments due to intricacy of subjective nature of human thoughts and easily express fuzzy information. Technique for order preference by similarity to an ideal solution (TOPSIS) is one of the eminent traditional distance measure-based approach for multi-criteria group decision-making (MCGDM) problems and having widespread applications. This study aims to develop TOPSIS method for MCGDM problems under IVIUL environment. Firstly, some basic operational laws and aggregation operators of IVIULS are discussed. A novel distance measure of IVIULEs is also investigated. An illustrative example of evaluation problem is also taken to clarify developed methodology and to reveal its efficiency with comparative analysis of proposed method.

In this present study, we have addressed the issue of hesitancy in fuzzy set based forecasting method that occurs due to the availability of several fuzzification method of time series data and propose integrated fuzzy time series forecasting method with hesitant fuzzy sets. In this study, partition the universe of discourse and used triangular and Gaussian fuzzy membership function to construct hesitant fuzzy sets. Further to this hesitant fuzzy element are aggregated by a suitable aggregation operator to construct fuzzy logical relations. Proposed forecasting method is implemented over the historical enrollments of Alabama University. The outperformance is tested over other forecasted models under the error measure, validation test and statistical analysis.

CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells. Second, Cas9 can be induced uniformly in the clonal cultures using doxycycline to measure the knockout phenotype. Third, two genes can be simultaneously knocked out using this approach. Finally, by not involving lentiviruses, our method is appealing to a broad research audience. Using this methodology we generated an inducible AGO2-knockout cell line showing normal RNA interference in the absence of doxycycline. Upon induction of Cas9, the AGO2 locus was cleaved, the AGO2 protein was depleted, and RNA interference was compromised. In addition to generating inducible knockouts, our technology can be adapted to improve other applications of Cas9, including transcriptional/epigenetic modulation and visualization of cellular DNA loci.

Telomerase replicates chromosome ends to facilitate continued cell division. Mutations that compromise telomerase function result in stem cell failure diseases, such as dyskeratosis congenita (DC). One such mutation (K170Δ), residing in the telomerase-recruitment factor TPP1, provides an excellent opportunity to structurally, biochemically, and genetically dissect the mechanism of such diseases. We show through site-directed mutagenesis and X-ray crystallography that this TPP1 disease mutation deforms the conformation of two critical amino acids of the TEL [TPP1’s glutamate (E) and leucine-rich (L)] patch, the surface of TPP1 that binds telomerase. Using CRISPR-Cas9 technology, we demonstrate that introduction of this mutation in a heterozygous manner is sufficient to shorten telomeres in human cells. Our findings rule out dominant-negative effects of the mutation. Instead, these findings implicate reduced TEL patch dosage in causing telomere shortening. Our studies provide mechanistic insight into telomerase-deficiency diseases and encourage the development of gene therapies to counter such diseases.

Background: Organotypic culture models developed using 3D conditions recapitulate tissue-specific structural features and cell-cell interactions more accurately than conventional 2D cultures. Our ultimate goal is to optimize culture conditions which promote the survival and proliferation of multiple myeloma (MM) cells and could serve as a platform for molecular mechanistic, clinical biomarker and pharmacodynamic marker studies using immune-modulatory compounds (IMIDs) and other myeloma drugs alone and in combination. Design/Results: Using gas permeable microfluidic devices, we cultured and compared growth/morphologic properties of six multiple myeloma cell lines, MM1.S, MM1.SPR, H929, H929PR, H929-220R and RPMI-8226 in 2D and 3D conditions. Collagen type IV was used as an extra-cellular matrix source to grow these cells. Cell growth and morphology was captured at regular intervals. Ten days post culture, cells were harvested from the device and stained for proliferation (Ki67 staining) index and expression of key MM oncogenic molecules, CD138, CD38 and BCMA. Cell lines grown in 3D conditions had, with some exceptions, higher proliferation index compared to 2D conditions. Thus, Ki67-mean fluorescence intensity (MFI) for 3D vs 2D were: 2038 vs 1130 for MM1.S; 1614 vs 1912 for MM1.PR; 2067 vs 1169 for H929; 2057 vs 1702 for H929PR; 2300 vs 1889 for H929-220R; 2018 vs 1220 for RPMI-8226. Similar trends for higher proliferation under 3D conditions were observed for the CD138, CD38 and BCMA cell subsets. Expression of FOXM1, a potential marker of IMID resistance, was reduced in Pomalidomide sensitive non-synchronous cells compared to resistant cells, although a few clusters with higher FOXM1 expression were observed among sensitive cells. To further study the effects of other components of MM tumor micro-environment on Pomalidomide response, we optimized the culture conditions to co-culture MM cell lines with bone marrow stromal cells. The co-culture of bone marrow stromal cells, HS5 with MM cell line H929 protected Ikaros degradation induced by Pomalidomide. Interestingly, CD44 expression in H929 cells was upregulated in co-culture conditions with stromal cells. Future Directions: These culture conditions are currently being optimized to study the (1) drug effects in MM and immune cells alone and in combination and (2) use the co-culture derived cells for single cell level evaluation of genetic, transcriptomic or proteomic changes associated with drug treatment and (3) ultimately grow primary Myeloma cells in these conditions for ex vivo manipulation and downstream molecular and biological effects. Figure. Figure. Disclosures Ahsan: celgene: Employment, Equity Ownership. Jeyaraju:Celgene Corporation: Employment, Equity Ownership. Bisht:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corporation: Employment, Equity Ownership. Pierceall:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

Chromosomal aberrations are a hallmark of human cancers, with complex cytogenetic rearrangements leading to genetic changes permissive for cancer initiation and progression. Protection of Telomere 1 (POT1) is an essential component of the shelterin complex and functions to maintain chromosome stability by repressing the activation of aberrant DNA damage and repair responses at telomeres. Sporadic and familial mutations in the oligosaccharide-oligonucleotide (OB) folds of POT1 have been identified in many human cancers, but the mechanism underlying how hPOT1 mutations initiate tumorigenesis has remained unclear. Here we show that the human POT1's OB-folds are essential for the protection of newly replicated telomeres. Oncogenic mutations in hPOT1 OB-fold fail to bind to single-stranded telomeric DNA, eliciting a DNA damage response at telomeres that promote inappropriate chromosome fusions via the mutagenic alternative non-homologous end joining (A-NHEJ) pathway. hPOT1 mutations also result in telomere elongation and the formation of transplantable hematopoietic malignancies. Strikingly, conditional deletion of both mPot1a and p53 in mouse mammary epithelium resulted in development of highly invasive breast carcinomas and the formation of whole chromosomes containing massive arrays of telomeric fusions indicative of multiple breakage-fusion-bridge cycles. Our results reveal that hPOT1 OB-folds are required to protect and prevent newly replicated telomeres from engaging in A-NHEJ mediated fusions that would otherwise promote genome instability to fuel tumorigenesis.

Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) that participates in a broad range of cellular activities due to interaction with multiple binding partners. Tankyrase 1 recognizes a linear six-amino-acid degenerate motif and, hence, has hundreds of potential target proteins. Binding of partner proteins to tankyrase 1 usually results in their poly(ADP-ribosyl)ation (PARsylation) and can lead to ubiquitylation and proteasomal degradation. However, it is not known how tankyrase 1 PARP activity is regulated. Here we identify GDP-mannose 4,6-dehydratase (GMD) as a binding partner of tankyrase 1. GMD is a cytosolic protein required for the first step of fucose synthesis. We show that GMD is complexed to tankyrase 1 in the cytosol throughout interphase, but its association with tankyrase 1 is reduced upon entry into mitosis, when tankyrase 1 binds to its other partners TRF1 (at telomeres) and NuMA (at spindle poles). In contrast to other binding partners, GMD is not PARsylated by tankyrase 1. Indeed, we show that GMD inhibits tankyrase 1 PARP activity in vitro, dependent on the GMD tankyrase 1 binding motif. In vivo, depletion of GMD led to degradation of tankyrase 1, dependent on the catalytic PARP activity of tankyrase 1. We speculate that association of tankyrase 1 with GMD in the cytosol sequesters tankyrase 1 in an inactive stable form that can be tapped by other target proteins as needed.

Germline mutations in telomere biology genes cause dyskeratosis congenita (DC), an inherited bone marrow failure and cancer predisposition syndrome. DC is a clinically heterogeneous disorder diagnosed by the triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia; Hoyeraal-Hreidarsson syndrome (HH), a clinically severe variant of DC, also includes cerebellar hypoplasia, immunodeficiency, and intrauterine growth retardation. Approximately 70% of DC cases are associated with a germline mutation in one of nine genes, the products of which are all involved in telomere biology. Using exome sequencing, we identified mutations in Adrenocortical Dysplasia Homolog (ACD) (encoding TPP1), a component of the telomeric shelterin complex, in one family affected by HH. The proband inherited a deletion from his father and a missense mutation from his mother, resulting in extremely short telomeres and a severe clinical phenotype. Characterization of the mutations revealed that the single-amino-acid deletion affecting the TEL patch surface of the TPP1 protein significantly compromises both telomerase recruitment and processivity, while the missense mutation in the TIN2-binding region of TPP1 is not as clearly deleterious to TPP1 function. Our results emphasize the critical roles of the TEL patch in proper stem cell function and demonstrate that TPP1 is the second shelterin component (in addition to TIN2) to be implicated in DC.

Sister chromatid cohesion relies on cohesin, a complex comprised of a tri-partite ring and a peripheral subunit Scc3, which is found as two related isoforms SA1 and SA2 in vertebrates. There is a division of labor between the vertebrate cohesin complexes; SA1-cohesin is required at telomeres and SA2-cohesin at centromeres. Depletion of SA1 has dramatic consequences for telomere function and genome integrity, but the mechanism by which SA1-cohesin mediates cohesion at telomeres is not well understood. Here we dissect the individual contribution of SA1 and the ring subunits to telomere cohesion and show that telomeres rely heavily on SA1 and to a lesser extent on the ring for cohesion. Using chromatin immunoprecipitation we show that SA1 is highly enriched at telomeres, is decreased at mitosis when cohesion is resolved, and is increased when cohesion persists. Overexpression of SA1 alone was sufficient to induce cohesion at telomeres, independent of the cohesin ring and dependent on its unique (not found in SA2) amino terminal domain, which we show binds to telomeric DNA via an AT-hook motif. We suggest that a specialized cohesion mechanism may be required to accommodate the high level of DNA replication-associated repair at telomeres.

The inability of sodium antimony gluconate (SAG)-unresponsive kala-azar patients to clear Leishmania donovani (LD) infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs) typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as antimony-resistant (SbRLD) and antimony-sensitive (SbSLD) was compared in vitro. Unlike SbSLD, infection of DCs with SbRLD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. SbRLD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-κB pathways. In contrast, SbSLD failed to block activation of SAG (20 µg/ml)-induced PI3K/AKT pathway; which continued to stimulate NF-κB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with SbSLD also inhibited SAG (20 µg/ml)-induced activation of PI3K/AKT and NF-κB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 µg/ml. In contrast, SbRLD inhibited these SAG-induced events regardless of duration of DC exposure to SbRLD or dose of SAG. Interestingly, the inhibitory effects of isogenic SbSLD expressing ATP-binding cassette (ABC) transporter MRPA on SAG-induced leishmanicidal effects mimicked that of SbRLD to some extent, although antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-κB was found to transcriptionally regulate expression of murine γglutamylcysteine synthetase heavy-chain (mγGCShc) gene, presumably an important regulator of antimony resistance. Importantly, SbRLD but not SbSLD blocked SAG-induced mγGCS expression in DCs by preventing NF-κB binding to the mγGCShc promoter. Our findings demonstrate that SbRLD but not SbSLD prevents SAG-induced DC activation by suppressing a PI3K-dependent NF-κB pathway and provide the evidence for differential host-pathogen interaction mediated by SbRLD and SbSLD., Author Summary Kala-azar, a life-threatening parasitic disease caused by Leishmania donovani (LD), is widening its base in different parts of the world. Currently, there is no effective vaccine against kala-azar. The antimonial drugs like sodium antimony gluconate (SAG) have been the mainstay of therapy for this disease. Recently, due to the emergence of antimony-resistance in parasites, SAG often fails to cure kala-azar patients, which is compounding the disaster further. It is still unknown how infection with LD exhibiting antimony-resistant phenotype, in contrast to antimony-sensitive phenotype, is handled by the kala-azar patients upon SAG treatment. This demands an understanding of the nature of host immune responses against these two distinct categories of parasites. Accordingly, we compared the impact of infection with LD exhibiting antimony-resistant versus antimony-sensitive phenotype on dendritic cells (DCs). DCs upon activation/maturation initiate anti-leishmanial immunity. We showed that parasites with antimony-resistant but not antimony-sensitive phenotype prevented SAG-induced DC activation/maturation by blocking activation of NF-κB. The latter is a key signaling pathway regulating DC activation/maturation. Our studies for the first time provide both a cellular and molecular basis for differential response of host cells to parasite isolates with antimony-resistant and antimony-sensitive phenotype, which may influence the outcome of the disease.

Heterochromatin assembly in fission yeast is initiated by binding of Swi6/HP1 to the Lys-9-dimethylated H3 followed by spreading via cooperative recruitment of Swi6/HP1. Recruitment of Cohesin by Swi6/HP1 further stabilizes the heterochromatin structure and integrity. Subsequently, polyubiquitylation of Cut2 by anaphase-promoting complex-cyclosome (APC/C)-ubiquitin-protein isopeptide ligase (E3 ligase) followed by degradation of Cut2 releases Cut1, which cleaves the Rad21 subunit of Cohesin, facilitating sister chromatid separation during mitosis. Here, we demonstrate a surprising role of APC/C in assembly of heterochromatin and silencing at mating type, centromere, and ribosomal DNA loci. Coincidentally with the loss of silencing, recruitment of Swi6, H3-Lys-9-Me2, and Clr4 at dg-dh repeats at cen1 and the K region of mat locus is abrogated in mutants cut4, cut9, and nuc2. Surprisingly, both Cut4 and Cut9 are also highly enriched at these regions in wild type and depleted in swi6Δ mutant. Cut4 and Cut9 interact directly with Swi6/HP1 and Clr4, whereas the mutant Cut4 does not, suggesting that a direct physical interaction of APC subunits Cut4 and Cut9 with Swi6 and Clr4 is instrumental in heterochromatin assembly. The silencing defect in APC mutants is causally related to ubiquitylation activity of APC-E3 ligase. Like swi6 mutant, APC mutants are also defective in Cohesin recruitment and exhibit defects like lagging chromosomes, chromosome loss, and aberrant recombination in the mat region. In addition, APC mutants exhibit a bidirectional expression of dh repeats, suggesting a role in the RNA interference pathway. Thus, APC and heterochromatin proteins Swi6 and Clr4 play a mutually cooperative role in heterochromatin assembly, thereby ensuring chromosomal integrity, inheritance, and segregation during mitosis and meiosis.

Telomere length is regulated by a complex interplay of several factors, including telomerase, telomere-binding proteins, DNA replication machinery and recombination. In yeast, DNA polymerase α is required for de novo synthesis of telomeres from broken ends of DNA, and it also suppresses the elongation of normal telomeric repeats. Heterochromatin proteins Clr1–Clr4 and Swi6 and DNA polα organize heterochromatin structure at mating type, centromere, rDNA and telomere regions that are refractory to transcription and recombination in Schizosaccharomyces pombe. Here, we have addressed the role of heterochromatin structure in regulating the integrity and organization of telomeric regions. Here, we show that subtelomeric duplication and rearrangements occur in polα and heterochromatin mutants and find that some of the putative duplication events are dependent on the Rad50 pathway. Thus, our study shows a role of heterochromatin in maintaining the integrity of the subtelomeric regions by suppressing their recombination in Sz. pombe. Copyright © 2008 John Wiley & Sons, Ltd.