CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

Fully accessible fitness landscape of oncogene-negative lung adenocarcinoma.

Cancer genomes are almost invariably complex with genomic alterations cooperating during each step of carcinogenesis. In cancers that lack a single dominant oncogene mutation, cooperation between the inactivation of multiple tumor suppressor genes can drive tumor initiation and growth. Here, we shed light on how the sequential acquisition of genomic alterations generates oncogene-negative lung tumors. We couple tumor barcoding with combinatorial and multiplexed somatic genome editing to characterize the fitness landscapes of three tumor suppressor genes NF1, RASA1, and PTEN, the inactivation of which jointly drives oncogene-negative lung adenocarcinoma initiation and growth. The fitness landscape was surprisingly accessible, with each additional mutation leading to growth advantage. Furthermore, the fitness landscapes remained fully accessible across backgrounds with the inactivation of additional tumor suppressor genes. These results suggest that while predicting cancer evolution will be challenging, acquiring the multiple alterations that drive the growth of oncogene-negative tumors can be facilitated by the lack of constraints on mutational order.

Biocatalytic stereoselective synthesis of pyrrolidine-2,3-diones containing all-carbon quaternary stereocenters.

Highly functionalized pyrrolidine-2,3-diones can be synthesized efficiently and stereoselectively under mild conditions using a biocatalytic approach. The reaction led to the formation of new all-carbon quaternary stereocenters from Myceliophthora thermophila laccase (Novozym 51003) catalyzed oxidation of catechols to ortho-quinones and subsequent 1,4-addition with 3-hydroxy-1,5-dihydro-2H-pyrrol-2-ones. The reaction was conducted with various substituents on both reactants, resulting in 13 products in moderate to good yields (42-91%). The same 15 reactions were also tested with K3Fe(CN)6 as a catalyst, but here only one reaction resulted in a product (60% yield).

Biocatalytic synthesis of oxadiazole thioethers and evaluation of their antitumor activity.

A multicomponent enzyme-catalyzed process is suggested for the synthesis of a novel series of 1,3,4-oxadiazole thioether derivatives with yields ranging from 65 to 94%. Novozym 435, the immobilized form of Candida antarctica lipase B (CALB), was found to efficiently catalyze the reaction. The products were evaluated for antitumor activities against two cancer cell lines, HT-29 (human colorectal cancer cell) and HepG2 (human liver cancer cell), by MTT assays. Among them, two compounds exhibited higher antitumor activities, for both cell lines, compared to doxorubicin. In silico molecular docking and computational ADME analysis were performed to propose a mode of action for the anti-cancer activities and to predict drug-likeness, respectively.

Association of dietary and blood inflammatory indicators with depression, anxiety, and stress in adults with vitamin D deficiency.

BACKGROUND: There is growing evidence that vitamin D may be related to mental health. The aim of the current study was to investigate the association of dietary and blood inflammatory factors with mental health disorders in subjects with vitamin D deficiency, shedding further light on the complex interplay of these conditions. METHOD: In this cross-sectional study, 306 subjects completed the validated Depression, Anxiety, and Stress Scale questionnaire to evaluate their depression, anxiety, and stress scores. Dietary inflammatory index (DII) and healthy eating index (HEI) were calculated using a validated 65-item food frequency questionnaire. Blood samples were taken and vitamin D, cytokine, and hs-CRP levels were measured using enzyme-linked immunosorbent assay kits. Platelet to lymphocyte ratio (PLR) and neutrophil to lymphocyte ratio (NLR) were calculated using standard laboratory methods. RESULTS: The subjects were divided into two groups based on their vitamin D levels: a vitamin D < 20 µg/dl group (N = 257) and a vitamin D ≥ 20  µg/dl group (N = 49). Between group analysis revealed that only DII (p = 0.015), platelet (p = 0.04), and hs-CRP (p = 0.015) were significantly different. In adults with vitamin D levels below 20 µg/dl, NLR and DII were significantly higher in subjects with anxiety (p < 0.05), and this relationship remained significant only for NLR after adjusting for age and sex. Additionally, PLR and HEI were significantly different in depressed compared to non-depressed subjects, and this association remained significant only for HEI after adjusting for age and sex. CONCLUSION: In subjects with vitamin D deficiency, increased levels of PLR, NLR, and DII were associated with depression and anxiety, while HEI was negatively associated with depression. These associations were not found in subjects with vitamin D levels ≥20 µg/dl.

Molecular typing of enteroviruses and parechoviruses in acute flaccid paralysis patients in Iran in 2019.

Enteroviruses (EVs) and parechoviruses (PeVs) are among the viral pathogens that can cause acute flaccid paralysis (AFP). There is not sufficient information about direct detection of EVs and PeVs in AFP patients in Iran. The aim of this study was to conduct a one-year study for direct detection and molecular typing of EVs and PeVs from stool samples of AFP patients in Iran. One hundred stool samples from polio-negative AFP patients who were referred to the Iran National Polio Laboratory were randomly chosen and analyzed during 2019. A one-step TaqMan probe-based real-time RT-PCR assay targeting the 5'-untranslated region (5' -UTR) was used to screen for EVs and PeVs. All positive samples were genotyped by direct sequencing, targeting the VP1 region of the genome. In total, twelve (12%) and four (4%) stool samples from polio-negative AFP children were positive for EVs and PeVs, respectively. Sequence analysis revealed the presence of echovirus 2 (E2), echovirus 13 (E13), echovirus 25 (E25), echovirus 30 (E30), coxsackievirus A2 (CVA2), coxsackievirus A9 (CVA9), coxsackievirus A16 (CVA16), human enterovirus A76 (HEV-A76), and human parechovirus 1 (HPeV1) in children with AFP-like symptoms. Phylogenetic analysis showed that E2 strains clustered together with the strains circulating in the Netherlands during 2014, whereas the PeV strains belonged to different lineages. This study demonstrates that different EV types are associated with AFP cases in Iran. However, the frequency of association of PeVs with AFP cases appears to be low.

A multi-component approach for co-immobilization of lipases on silica-coated magnetic nanoparticles: improving biodiesel production from waste cooking oil.

The capability of multi-component reactions in rapid immobilization of enzymes was considered for co-immobilization of Thermomyces lanuginous lipase (TLL) and Candida antarctica lipase B (CALB) [TLL: CALB]; Rhizomucor miehei lipase (RML) and CALB [RML: CALB] on amine-functionalized silica-coated magnetic nanoparticles (Fe3O4@SiO2-NH2). Immobilization of different ratios of lipases was performed within 3 h under mild conditions; producing specific activity ranging from 29 to 35 U/mg for TLL:CALB and 21-34 U/mg for RML:CALB. The co-immobilized derivatives showed improved co-solvent and thermal stability compared to the corresponding free enzymes. All the derivatives were also used to catalyze the transesterification of waste cooking oil with methanol to produce biodiesel (fatty acid methyl esters). Response surface method (RSM) and a central composite rotatable design (CCRD) were used to study the effects of different factors on the FAME yield. Fe3O4@SiO2-NH2-RML-CALB and Fe3O4@SiO2-NH2-TLL-CALB had maximum FAME yields of 99-80%, respectively.

Structured light engineering using a photonic nanojet.

In this Letter, we present the photonic nanojet as a phenomenon in a structured light generator system that is implemented to modify the source focal spot size and emission angle. The optical system comprises a microlens array that is illuminated by a focused Gaussian beam to generate a structured pattern in the far field. By introducing a spheroid with different aspect ratios in the focus of the Gaussian beam, the source optical characteristics change, and a photonic nanojet is generated, which will engineer the far-field distribution. To probe the light fields, we implement a high-resolution interferometry setup to extract both the phase and intensity at different planes. We both numerically and experimentally demonstrate that the pattern distribution in the far field can be engineered by a photonic nanojet. As an example, we examine prolate, sphere, and oblate geometries. An interesting finding is that depending on the spheroid geometry, a smaller transverse FWHM of a photonic nanojet with a higher divergence angle produces an increased pattern field of view at the same physical size of the optical system.

Molecular characterization of non-polio enteroviruses isolated from children with acute flaccid paralysis in IRAN, 2015-2018.

In addition to polioviruses, non-polio enteroviruses (NPEVs) are frequently isolated from patients with acute flaccid paralysis (AFP) worldwide. In polio-free countries, there have been expectations that with disappearing wild poliovirus from the community, the rate of AFP would decrease, but the increasing number of AFP cases proved this notion to be wrong. There are speculations that NPEVs might be the cause of increasing AFP rate. The aim of this study was to investigate frequency, genetic diversity, circulation patterns of NPEVs isolated from AFP cases in Iran from 2015 to 2018. Fifty-three NPEVs were isolated from stool specimens of AFP cases during four years of AFP surveillance. Nested PCR and VP1 sequencing revealed 20 NPEV types in which Echovirus 3 (13.2%), Echovirus 6 (13.2%), Echovirus 7 (7.5%), Echovirus 13 (7.5%) and Echovirus 21 (7.5%) were the most frequent. Coxsackie B viruses were isolated for the first time in AFP cases in Iran. The phylogenetic analysis of Echovirus 3 and Echovirus 6 revealed that Iranian echovirus strains belonged to the same cluster, indicating these viruses have been circulating in Iran for a long time. Compared to global Echovirus 3 and Echovirus 6 references, Echovirus 3 and Echovirus 6 strains detected in this study were closely related to Indian and Malaysia strains, respectively. The results of this study demonstrated a wide variety of NPEV types in Iranian patients, some of which had not been reported in previous studies. Moreover, this study highlights the need for NPEV surveillance in AFP cases.

Porcine pancreas lipase as a green catalyst for synthesis of bis-4-hydroxy coumarins.

4-Hydroxycoumarins represents an important structural motif in life sciences molecules with remarkable biological properties. 4-Hydroxycoumarins are important precursors in the synthesis of organic compounds and critical pharmacophores in medicinal chemistry. One of the derivatives of this compound is biscoumarin, which has anticoagulant medicinal properties. Porcine pancreas lipase (PPL) has been used as an efficient green biocatalyst for the synthesis of bis-4-hydroxycoumarin compounds and expands the biocatalytic promiscuity of lipase in organic synthesis. Some aromatic aldehydes have been used in this investigation for screening and examining the ability of enzyme in knoevenagel condensation. Various parameters including temperature, solvent, and the amount of biocatalyst were investigated, ethanol, 45 °C and 10 mg of PPL gave rise to best yields. Bis-4-hydroxy coumarin compounds were synthesized under mild reaction conditions with high conversion yields (81-88%).

Fine Modulation of the Catalytic Properties of Rhizomucor miehei Lipase Driven by Different Immobilization Strategies for the Selective Hydrolysis of Fish Oil.

Functional properties of each enzyme strictly depend on immobilization protocol used for linking enzyme and carrier. Different strategies were applied to prepare the immobilized derivatives of Rhizomucor miehei lipase (RML) and chemically aminated RML (NH2-RML). Both RML and NH2-RML forms were covalently immobilized on glyoxyl sepharose (Gx-RML and Gx-NH2-RML), glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH2-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH2-RML) and heterofunctional epoxy support partially modified with iminodiacetic acid (epoxy-IDA-RML and epoxy-IDA-NH2-RML). Immobilization varied from 11% up to 88% yields producing specific activities ranging from 0.5 up to 1.9 UI/mg. Great improvement in thermal stability for Gx-DTT-NH2-RML and epoxy-IDA-NH2-RML derivatives was obtained by retaining 49% and 37% of their initial activities at 70 °C, respectively. The regioselectivity of each derivative was also examined in hydrolysis of fish oil at three different conditions. All the derivatives were selective between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. The highest selectivity (32.9 folds) was observed for epoxy-IDA-NH2-RML derivative in the hydrolysis reaction performed at pH 5 and 4 °C. Recyclability study showed good capability of the immobilized biocatalysts to be used repeatedly, retaining 50-91% of their initial activities after five cycles of the reaction.

Vaccine-Derived Poliovirus Infection among Patients with Primary Immunodeficiency and Effect of Patient Screening on Disease Outcomes, Iran.

Patients with immunodeficiency-associated vaccine-derived poliovirus (iVDPV) are potential poliovirus reservoirs in the posteradication era that might reintroduce polioviruses into the community. We update the iVDPV registry in Iran by reporting 9 new patients. In addition to national acute flaccid paralysis surveillance, cases were identified by screening nonparalyzed primary immunodeficiency (PID) patients. Overall, 23 iVDPV patients have been identified since 1995. Seven patients (30%) never had paralysis. Poliovirus screening accelerated the iVDPV detection rate in Iran after 2014.The iVDPV infection rate among nonparalyzed patients with adaptive PID was 3.1% (7/224), several folds higher than previous estimates. Severe combined immunodeficiency patients had the highest risk for asymptomatic infection (28.6%) compared with other PIDs. iVDPV2 emergence has decreased after the switch from trivalent to bivalent oral poliovirus vaccine in 2016. However, emergence of iVDPV1 and iVDPV3 continued. Poliovirus screening in PID patients is an essential step in the endgame of polio eradication.

Piperazine: An excellent catalyst for the synthesis of 2-amino-3-cyano-4H-pyrans derivatives in aqueous medium.

An improved, forthright, and highly efficient one-pot synthesis of a wide range of pharmaceutically exciting diverse kind of functionalized 2-amino-3-cyano-4H-pyrans and especially bis-pyrans compounds is developed using piperazine as a low-cost and environmentally benign commercially available basic catalyst in aqueous media. The attractive features of this process are simple procedure, short reaction times, high yields, no column chromatographic separation and commercial availability and recyclability of the catalyst. Also, piperazine can catalyze the synthesis of the target compounds on a larger scale. Furthermore, rational mechanism was suggested via GC-Mass analysis of the trapped intermediates.

Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1).

Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production.

Blunt Blade Subcision: An Evolution in the Treatment of Atrophic Acne Scars.

BACKGROUND: Subcision is the mainstay of atrophic acne scar treatment but the efficacy and safety of the procedure is controversial. OBJECTIVE: To improve the efficacy of the subcision procedure, a blunt subcision blade was designed and evaluated. METHODS: Eighteen patients with bilateral atrophic acne scars considered eligible for subcision were enrolled. Before subcision, a tumescent solution was injected subdermally to anesthetize the treatment area and aid the dissection of the dermal-subcutaneous tissue. Patients underwent treatment using the blunt subcision blade, a long metal blade with gradually narrowing edges, and a rounded blunt tip. Early postoperative complications, overall aesthetic improvement and persistent discoloration, or lumpiness were assessed 7 days after subcision and at a 6-month follow-up visit. RESULTS: Moderate to marked improvement of atrophic scars was observed in 15 cases (83.3%). Mild to moderate tenderness, periorbital ecchymoses, and swelling were reported by some patients, but resolved completely within 1 week after the procedure. No cases of persistent discoloration or lumpiness were observed at the final visit. CONCLUSION: The blunt blade subcision procedure is suggested as an effective method for the treatment of atrophic acne scars.

Heterogeneity in readouts of canonical wnt pathway activity within intestinal crypts.

BACKGROUND: Canonical Wnt pathway signaling is necessary for maintaining the proliferative capacity of mammalian intestinal crypt base columnar stem cells (CBCs). Furthermore, dysregulation of the Wnt pathway is a major contributor to disease, including oncogenic transformation of the intestinal epithelium. Given the critical importance of this pathway, numerous tools have been used as proxy measures for Wnt pathway activity, yet the relationship between Wnt target gene expression and reporter allele activity within individual cells at the crypt base remains unclear. RESULTS: Here, we describe a novel Axin2-CreERT2-tdTomato allele that efficiently marks both Wnt(High) CBCs and radioresistant reserve intestinal stem cells. We analyze the molecular and functional identity of Axin2-CreERT2-tdTomato-marked cells using single cell gene expression profiling and tissue regeneration assays and find that Axin2 reporter activity does not necessarily correlate with expression of Wnt target genes and, furthermore, that Wnt target genes themselves vary in their expression patterns at the crypt base. CONCLUSIONS: Wnt target genes and reporter alleles can vary greatly in their cell-type specificity, demonstrating that these proxies cannot be used interchangeably. Furthermore, Axin2-CreERT2-tdTomato is a robust marker of both active and reserve intestinal stem cells and is thus useful for understanding the intestinal stem cell compartment. Developmental Dynamics 245:822-833, 2016. © 2016 Wiley Periodicals, Inc.

Vaccine-Derived Polioviruses and Children with Primary Immunodeficiency, Iran, 1995-2014.

Widespread use of oral poliovirus vaccine has led to an ≈99.9% decrease in global incidence of poliomyelitis (from ≈350,000 cases in 1988 to 74 cases in 2015) and eradication of wild-type poliovirus serotypes 2 and 3. However, patients with primary immunodeficiency might shed vaccine-derived polioviruses (VDPVs) for an extended period, which could pose a major threat to polio eradication programs. Since 1995, sixteen VDPV populations have been isolated from 14 patients with immunodeficiency in Iran. For these patients, vaccine-associated paralysis, mostly in >1 extremity, was the first manifestation of primary immunodeficiency. Seven patients with humoral immunodeficiency cleared VDPV infection more frequently than did 6 patients with combined immunodeficiencies. Our results raise questions about manifestations of VDPVs in immunodeficient patients and the role of cellular immunity against enterovirus infections. On the basis of an association between VDPVs and immunodeficiency, we advocate screening of patients with primary immunodeficiency for shedding of polioviruses.

No evidence of mammary tumor virus env gene-like sequences among Iranian women with breast cancer.

OBJECTIVE: The mouse mammary tumor virus (MMTV) is the well-established etiological agent of mammary tumors in mice. A series of studies have implicated that a human murine mammary tumor virus-like virus occurs in human breast cancer, but it is unclear whether it has any causal role. METHODS: The aim of the present study was to investigate the presence of MMTV env gene-like sequences in a group of Iranian women with or without breast cancer. A total of 65 breast cancer and 65 noncancerous breast specimens from the Department of Pathology of Tabriz University in East Azerbaijan, Iran, were analyzed by nested PCR. RESULTS: All breast cancer and benign breast samples were negative for MMTV env gene-like DNA. CONCLUSION: These results indicate that the MMTV env gene-like virus may not play a significant role in the etiology of breast cancer among Iranian women.

The effect of carbon chain length of cross-linking agent on the functionality of carrier- free immobilized Thermomyces lanuginosa lipase particles.

The cross-linked enzyme (CLEs) of Thermomyces lanuginosa lipase (TLL) was prepared in an isocyanide-based multi-component reactions (ICMRs) platform by applying three di-acidic cross-linkers to unveil more factors contributing to the functional properties of CLEs. The linkers were 1,11-undecanedicarboxylic acid, azelaic acid, and adipic acid with 11, 7, and 4 carbon lengths, respectively, providing a proper tool to investigate the effect of linker length on the activity, stability, and selectivity of the resulting CLEs. The immobilization yields of 60-90 % and the specific activities of 168, 88.4 and 49 U/mg were obtained for the CLEs of 1,11-undecanedicarboxylic acid, azelaic acid, adipic acid, respectively. The lower activity of azelaic and adipic acid-mediated CLEs compared to the soluble TLL (110 U/mg) was explained by in silico calculations. The results revealed that as opposed to 1,11-undecanedicarboxylic acid, both linkers tended to penetrate the enzyme active site, thus resulting in a major inhibitory effect on the enzyme functionality. The thermal and co-solvent stability of the immobilized derivatives improved compared to those of free TLL. The selectivity of CLEs was also examined by catalytic release of main omega-3 fatty acids from fish oil, presenting the highest selectivity of 22 for the CLEs of azelaic acid.

Exploring biodiversity patterns at the landscape scale by linking landscape energy and land use/land cover heterogeneity.

Agricultural Biodiversity dynamics has been evaluated by social metabolism or by landscape structure-function analysis. In this study, by using ELIA modeling, we used both methods in combination to understand how the interplay between social metabolism and landscape structure-function can affect biodiversity pattern distribution. We used energy reinvestment (E) as an indicator of social metabolism and landscape heterogeneity (Le) as an indicator of landscape structure-function. We propose a research hypothesis to analyze biodiversity patterns considering four different clusters identified based on high or low E or Le. As cluster 1, we defined E as high and Le as low and associated natural ecosystems to it. These ecosystems are expected to contain high species abundance but low richness. As cluster 2, both E and Le were defined as high and semi-natural ecosystems were associated to it, where nature friendly farm system developed. In these ecosystems, high species abundance and richness are expected. Cluster 3 with low E and Le was associated intensive farmland, which is due to the simplification of the landscape. Here, low energy reinvestment and landscape heterogeneity confirm that ecosystem services related to biodiversity have been drastically reduced. Lastly, cluster 4 with low E but high Le refers to intensive mosaics of farmland and pasture. In this cluster, the biodiversity richness index is high due to spatial landscape diversity, but the biodiversity abundance index is low due to the lack of energy reinvestment. We evaluate the proposed hypothesis for biodiversity analysis in the Qazvin province, emphasizing the interplay between energy availability and landscape heterogeneity in shaping ecological communities. This study highlights the importance of understanding biodiversity patterns at spatial scale and emphasizes the need for interdisciplinary research to address conservation and sustainability challenges. Our approach would be very useful where there is lack of biodiversity data.