High-throughput tagging of endogenous loci for rapid characterization of protein function.

To facilitate the interrogation of protein function at scale, we have developed high-throughput insertion of tags across the genome (HITAG). HITAG enables users to rapidly produce libraries of cells, each with a different protein of interest C-terminally tagged. HITAG is based on a modified strategy for performing Cas9-based targeted insertions, coupled with an improved approach for selecting properly tagged lines. Analysis of the resulting clones generated by HITAG reveals high tagging specificity, with most successful tagging events being indel free. Using HITAG, we fuse mCherry to a set of 167 stress granule-associated proteins and elucidate the features that drive a subset of proteins to strongly accumulate within these transient RNA-protein granules.

Controlled experimental generation of perturbed high-order Ince-Gaussian laser modes.

We present an experimental approach for generating perturbed high-order Ince-Gaussian laser modes by transforming the low and moderate-intensity lobes of high-order Ince-Gaussian (IG) modes into high-intensity lobes and vice versa. This perturbation reshuffles optical energy among the different lobes and generates new, to the best of our knowledge, modulated Ince-Gaussian (MIG) modes. Computer-generated holograms displayed over spatial light modulators were used to modulate the IGMs. Compared to IG modes, MIG modes are generated precisely in a sense that enhances the IG modes and provides a maximum number of highly intense lobes in a particular mode. That enables the newly generated MIG modes to be utilized more efficiently than IG modes in applications such as particle manipulation and optical trapping of microparticles, which exploit highly intense lobes.

Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis.

Bone marrow vascular endothelial cells (BM EC) regulate multiple myeloma pathogenesis. Identification of the mechanisms underlying this interaction could lead to the development of improved strategies for treating multiple myeloma. Here, we performed a transcriptomic analysis of human ECs with high capacity to promote multiple myeloma growth, revealing overexpression of the receptor tyrosine kinases, EPHB1 and EPHB4, in multiple myeloma-supportive ECs. Expression of ephrin B2 (EFNB2), the binding partner for EPHB1 and EPHB4, was significantly increased in multiple myeloma cells. Silencing EPHB1 or EPHB4 in ECs suppressed multiple myeloma growth in coculture. Similarly, loss of EFNB2 in multiple myeloma cells blocked multiple myeloma proliferation and survival in vitro, abrogated multiple myeloma engraftment in immune-deficient mice, and increased multiple myeloma sensitivity to chemotherapy. Administration of an EFNB2-targeted single-chain variable fragment also suppressed multiple myeloma growth in vivo. In contrast, overexpression of EFNB2 in multiple myeloma cells increased STAT5 activation, increased multiple myeloma cell survival and proliferation, and decreased multiple myeloma sensitivity to chemotherapy. Conversely, expression of mutant EFNB2 lacking reverse signaling capacity in multiple myeloma cells increased multiple myeloma cell death and sensitivity to chemotherapy and abolished multiple myeloma growth in vivo. Complementary analysis of multiple myeloma patient data revealed that increased EFNB2 expression is associated with adverse-risk disease and decreased survival. This study suggests that EFNB2 reverse signaling controls multiple myeloma pathogenesis and can be therapeutically targeted to improve multiple myeloma outcomes. SIGNIFICANCE: Ephrin B2 reverse signaling mediated by endothelial cells directly regulates multiple myeloma progression and treatment resistance, which can be overcome through targeted inhibition of ephrin B2 to abolish myeloma.

Super-oscillatory spots with different inhomogeneous linear polarized states.

We present the formation of super-oscillatory (SO) spots by tightly focusing the inhomogeneous linear polarized beam of different polarization states. At the entrance pupil of the focusing lens, a suitable phase manipulation in the incident beam results in a small super-oscillatory spot. Our numerical study based on the vectorial diffraction theory shows that SO spots of controllable size and various polarization combinations are possible. We also discuss the effect of the different polarization patterns of the incident beam on the size and energy distribution of the generated SO spots, which are potentially valuable for the orientation determination of single molecules and polarization-resolved imaging. This study reveals more influence of polarization states on the different components of the focused beam under the utilization of the proposed method rather than the usual tight focusing conditions.

Controllable experimental modulation of high-order Laguerre-Gaussian laser modes.

High-order helical and sinusoidal Laguerre-Gaussian (LG) laser modes have uneven energy distribution among their multiple concentric vortex core rings and lobes, respectively. Here, we explore an experimental method to reshuffle the optical energy among their multiple concentric vortex core rings and lobes of high-order LG modes in a controllable manner. We numerically designed a diffractive optical element displayed over a spatial light modulator to rearrange optical energy among multiple concentric vortex core rings. This changes outer low-intensity concentric vortex core rings into high-intensity vortex core rings of high-order helical LG modes at the Fourier plane. The precise generation of a high-order modulated helical LG laser mode has a maximum number of highly intense concentric vortex core rings compared to known standard helical LG modes. Further, this method is extended to high-order sinusoidal LG modes consisting of both low- and high-intensity lobes to realize modulated sinusoidal LG modes with a maximum number of highly intense lobes in a controllable manner. We envisage that the modulated helical and sinusoidal high-order LG modes may surpass standard LG modes in many applications where highly intense rings and lobes are crucial, as in particle manipulation of micro- and nanoparticles, and optical lithography.

Recurrent Desmoid Fibromatosis of the Thyroid Gland: A Diagnostic Challenge.

A 31-year-old Indian female with a history of near-total thyroidectomy 2.5 years prior presented with recurrent neck swelling. Magnetic resonance imaging (MRI) of the neck revealed an infiltrating mass involving the thyroid bed. Biopsy from the mass and review of slides from the previous thyroidectomy revealed a spindle cell tumour with interspersed areas of fibrosis and infiltrative edges entrapping thyroid follicles. Beta-catenin immunopositivity and CTNNB1 mutation confirmed the diagnosis of fibromatosis. The case is being reported for its rarity and the discussion of its differential diagnoses.

Nicotinamide riboside rescues dysregulated glycolysis and fatty acid ß-oxidation in a human hepatic cell model of citrin deficiency.

Citrin deficiency (CD) is an inborn error of metabolism caused by loss-of-function of the mitochondrial aspartate/glutamate transporter, CITRIN, which is involved in both the urea cycle and malate-aspartate shuttle. Patients with CD develop hepatosteatosis and hyperammonemia but there is no effective therapy for CD. Currently, there are no animal models that faithfully recapitulate the human CD phenotype. Accordingly, we generated a CITRIN knockout HepG2 cell line using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 genome editing technology to study metabolic and cell signaling defects in CD. CITRIN KO cells showed increased ammonia accumulation, higher cytosolic ratio of reduced versus oxidized form of nicotinamide adenine dinucleotide (NAD) and reduced glycolysis. Surprisingly, these cells showed impaired fatty acid metabolism and mitochondrial activity. CITRIN KO cells also displayed increased cholesterol and bile acid metabolism resembling those observed in CD patients. Remarkably, normalizing cytosolic NADH:NAD+ ratio by nicotinamide riboside increased glycolysis and fatty acid oxidation but had no effect on the hyperammonemia suggesting the urea cycle defect was independent of the aspartate/malate shuttle defect of CD. The correction of glycolysis and fatty acid metabolism defects in CITRIN KO cells by reducing cytoplasmic NADH:NAD+ levels suggests this may be a novel strategy to treat some of the metabolic defects of CD and other mitochondrial diseases.

Creation of pure longitudinal super-oscillatory spot.

We present a method that creates a super-oscillatory focal spot of a tightly focused radially polarized beam using the concept of a phase mask. Using vector diffraction theory, we report a super-oscillatory focal spot that is much smaller than the diffraction limit and the super-oscillation criterion. The proposed mask works as a special polarization filter that enhances the longitudinal component and filters out the transverse component of radial polarization at focus, permitting the creation of a pure longitudinal super-oscillatory focal spot.

Metabolic switching of estrogen-related receptor alpha in breast cancer aggression.

Orphan nuclear receptor estrogen-related receptor alpha (ERRα) is an important regulator of energy metabolism, whereas its hyperactivation in breast cancer has been shown to regulate cell migration, proliferation, and tumour development. These findings suggest a fine balance in the status of ERRα in regulating metabolic homeostasis or promoting cancer progression. In this issue, Brindisi et al. have shown that ERRα is endogenously activated by cholesterol and caused breast cancer aggressiveness. This study also supports the anti-tumour mechanisms of cholesterol-lowering drugs such as statins.

Experimental realization of modulated Hermite-Gaussian laser modes: a maximum number of highly intense lobes.

Here, we present an experimental method that redistributes the optical energy among the lobes of high-order standard Hermite-Gaussian (SHG) laser modes in a controlled manner. We numerically designed diffractive optical elements, displayed over a spatial light modulator for redistribution of optical energy that converts low and moderate intense lobes into all highly intense lobes and vice versa at the Fourier plane. Such precise generation of modulated HG (MHG) laser modes offers a maximum number of highly intense lobes compared to SHG modes. Hence, we envisage that MHG beams may surpass SHG beams in many applications, such as particle manipulation and optical lithography, where highly intense lobes play a significant role.

Spermidine-mediated hypusination of translation factor EIF5A improves mitochondrial fatty acid oxidation and prevents non-alcoholic steatohepatitis progression.

Spermidine is a natural polyamine that has health benefits and extends life span in several species. Deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) are key enzymes that utilize spermidine to catalyze the post-translational hypusination of the translation factor EIF5A (EIF5AH). Here, we have found that hepatic DOHH mRNA expression is decreased in patients and mice with non-alcoholic steatohepatitis (NASH), and hepatic cells treated with fatty acids. The mouse and cell culture models of NASH have concomitant decreases in Eif5aH and mitochondrial protein synthesis which leads to lower mitochondrial activity and fatty acid ß-oxidation. Spermidine treatment restores EIF5AH, partially restores protein synthesis and mitochondrial function in NASH, and prevents NASH progression in vivo. Thus, the disrupted DHPS-DOHH-EIF5AH pathway during NASH represents a therapeutic target to increase hepatic protein synthesis and mitochondrial fatty acid oxidation (FAO) and prevent NASH progression.

Gastric Outlet Obstruction Following Recurrent Pancreatitis Uncovers a Giant Parathyroid Adenoma: A Case Report.

A 35-year-old female presented with abdominal pain, fever, projectile vomiting, and a diffuse tender epigastric mass. She was diagnosed to have acute persistent pancreatitis with a pancreatic pseudocyst. Elevated serum calcium levels provided an etiologic link between hypercalcemia and pancreatitis. On examination, a nodule was found in the left side of her neck which was later diagnosed as a giant left inferior parathyroid adenoma. This report highlights the critical analysis of history, examination, and investigations to reach an ultimate diagnosis. Pseudocyst drainage and parathyroidectomy resolved her symptoms.

Vitamin B12 and folate decrease inflammation and fibrosis in NASH by preventing syntaxin 17 homocysteinylation.

BACKGROUND & AIMS: Several recent clinical studies have shown that serum homocysteine (Hcy) levels are positively correlated, while vitamin B12 (B12) and folate levels are negative correlated, with non-alcoholic steatohepatitis (NASH) severity. However, it is not known whether hyperhomocysteinemia (HHcy) plays a pathogenic role in NASH. METHODS: We examined the effects of HHcy on NASH progression, metabolism, and autophagy in dietary and genetic mouse models, patients, and primates. We employed vitamin B12 (B12) and folate (Fol) to reverse NASH features in mice and cell culture. RESULTS: Serum Hcy correlated with hepatic inflammation and fibrosis in NASH. Elevated hepatic Hcy induced and exacerbated NASH. Gene expression of hepatic Hcy-metabolizing enzymes was downregulated in NASH. Surprisingly, we found increased homocysteinylation (Hcy-lation) and ubiquitination of multiple hepatic proteins in NASH including the key autophagosome/lysosome fusion protein, Syntaxin 17 (Stx17). This protein was Hcy-lated and ubiquitinated, and its degradation led to a block in autophagy. Genetic manipulation of Stx17 revealed its critical role in regulating autophagy, inflammation and fibrosis during HHcy. Remarkably, dietary B12/Fol, which promotes enzymatic conversion of Hcy to methionine, decreased HHcy and hepatic Hcy-lated protein levels, restored Stx17 expression and autophagy, stimulated ß -oxidation of fatty acids, and improved hepatic histology in mice with pre-established NASH. CONCLUSIONS: HHcy plays a key role in the pathogenesis of NASH via Stx17 homocysteinylation. B12/folate also may represent a novel first-line therapy for NASH. LAY SUMMARY: The incidence of non-alcoholic steatohepatitis, for which there are no approved pharmacological therapies, is increasing, posing a significant healthcare challenge. Herein, based on studies in mice, primates and humans, we found that dietary supplementation with vitamin B12 and folate could have therapeutic potential for the prevention or treatment of non-alcoholic steatohepatitis.

Population Structure Analysis and Association Mapping for Turcicum Leaf Blight Resistance in Tropical Maize Using SSR Markers.

Maize is an important cereal crop in the world for feed, food, fodder, and raw materials of industries. Turcicum leaf blight (TLB) is a major foliar disease that can cause more than 50% yield losses in maize. Considering this, the molecular diversity, population structure, and genome-wide association study (GWAS) for TLB resistance were studied in 288 diverse inbred lines genotyped using 89 polymorphic simple sequence repeats (SSR) markers. These lines werescreened for TLB disease at two hot-spot locations under artificially inoculated conditions. The average percent disease incidence (PDI) calculated for each genotype ranged from 17 (UMI 1201) to 78% (IML 12-22) with an overall mean of 40%. The numbers of alleles detected at a locus ranged from twoto nine, with a total of 388 alleles. The polymorphic information content (PIC) of each marker ranged between 0.04 and 0.86. Out of 89 markers, 47 markers were highly polymorphic (PIC ≥ 0.60). This indicated that the SSR markers used were very informative and suitable for genetic diversity, population structure, and marker-trait association studies.The overall observed homozygosity for highly polymorphic markers was 0.98, which indicated that lines used were genetically pure. Neighbor-joining clustering, factorial analysis, and population structure studies clustered the 288 lines into 3-5 groups. The patterns of grouping were in agreement with the origin and pedigree records of the genotypesto a greater extent.A total of 94.10% lines were successfully assigned to one or another group at a membership probability of ≥0.60. An analysis of molecular variance (AMOVA) revealed highly significant differences among populations and within individuals. Linkage disequilibrium for r2 and D' between loci ranged from 0 to 0.77 and 0 to 1, respectively. A marker trait association analysis carried out using a general linear model (GLM) and mixed linear model (MLM), identified 15 SSRs markers significantly associated with TLB resistance.These 15 markers were located on almost all chromosomes (Chr) except 7, 8, and 9. The phenotypic variation explained by these loci ranged from 6% (umc1367) to 26% (nc130, phi085). Maximum 7 associated markers were located together on Chr 2 and 5. The selected regions identified on Chr 2 and 5 corroborated the previous studies carried out in the Indian maize germplasm. Further, 11 candidate genes were identified to be associated with significant markers. The identified sources for TLB resistance and associated markers may be utilized in molecular breeding for the development of suitable genotypes.

Genetic Diversity, Population Structure and Linkage Disequilibrium Analyses in Tropical Maize Using Genotyping by Sequencing.

Several maize breeding programs in India have developed numerous inbred lines but the lines have not been characterized using high-density molecular markers. Here, we studied the molecular diversity, population structure, and linkage disequilibrium (LD) patterns in a panel of 314 tropical normal corn, two sweet corn, and six popcorn inbred lines developed by 17 research centers in India, and 62 normal corn from the International Maize and Wheat Improvement Center (CIMMYT). The 384 inbred lines were genotyped with 60,227 polymorphic single nucleotide polymorphisms (SNPs). Most of the pair-wise relative kinship coefficients (58.5%) were equal or close to 0, which suggests the lack of redundancy in the genomic composition in the majority of inbred lines. Genetic distance among most pairs of lines (98.3%) varied from 0.20 to 0.34 as compared with just 1.7% of the pairs of lines that differed by <0.20, which suggests greater genetic variation even among sister lines. The overall average of 17% heterogeneity was observed in the panel indicated the need for further inbreeding in the high heterogeneous genotypes. The mean nucleotide diversity and frequency of polymorphic sites observed in the panel were 0.28 and 0.02, respectively. The model-based population structure, principal component analysis, and phylogenetic analysis revealed three to six groups with no clear patterns of clustering by centers-wise breeding lines, types of corn, kernel characteristics, maturity, plant height, and ear placement. However, genotypes were grouped partially based on their source germplasm from where they derived.

Thyroid Hormone Decreases Hepatic Steatosis, Inflammation, and Fibrosis in a Dietary Mouse Model of Nonalcoholic Steatohepatitis.

Background: Nonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, lobular inflammation, and fibrosis. Thyroid hormone (TH) reduces steatosis; however, the therapeutic effect of TH on NASH-associated inflammation and fibrosis is not known. This study examined the therapeutic effect of TH on hepatic inflammation and fibrosis during NASH and investigated THs molecular actions on autophagy and mitochondrial biogenesis. Methods: HepG2-TRß cells were treated with bovine serum albumin-conjugated palmitic acid (PA) to mimic lipotoxic conditions in vitro. Mice with NASH were established by feeding C57BL/6J mice Western diet with 15% fructose in drinking water for 16 weeks. These mice were administered triiodothyronine (T3)/thyroxine (T4) supplemented in drinking water for the next eight weeks. Results: In cultured HepG2-TRß cells, TH treatment increased mitochondrial respiration and fatty acid oxidation under basal and PA-treated conditions, as well as decreased lipopolysaccharides and PA-stimulated inflammatory and fibrotic responses. In a dietary mouse model of NASH, TH administration decreased hepatic triglyceride content (3.19 ± 0.68 vs. 8.04 ± 0.42 mM/g liver) and hydroxyproline (1.44 ± 0.07 vs. 2.58 ± 0.30 mg/g liver) when compared with mice with untreated NASH. Metabolomics profiling of lipid metabolites showed that mice with NASH had increased triacylglycerol, diacylglycerol, monoacylglycerol, and hepatic cholesterol esters species, and these lipid species were decreased by TH treatment. Mice with NASH also showed decreased autophagic degradation as evidenced by decreased transcription Factor EB and lysosomal protease expression, and accumulation of LC3B-II and p62. TH treatment restored the level of lysosomal proteins and resolved the accumulation of LC3B-II and p62. Impaired mitochondrial biogenesis was also restored by TH. The simultaneous restoration of autophagy and mitochondrial biogenesis by TH increased ß-oxidation of fatty acids. Additionally, the elevated oxidative stress and inflammasome activation in NASH liver were also decreased by TH. Conclusions: In a mouse model of NASH, TH restored autophagy and mitochondrial biogenesis to increase ß-oxidation of fatty acids and to reduce lipotoxicity, oxidative stress, hepatic inflammation, and fibrosis. Activating thyroid hormone receptor in the liver may represent an effective strategy for NASH treatment.

Caffeine prevents restenosis and inhibits vascular smooth muscle cell proliferation through the induction of autophagy.

Caffeine is among the most highly consumed substances worldwide, and it has been associated with decreased cardiovascular risk. Although caffeine has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), the mechanism underlying this effect is unknown. Here, we demonstrated that caffeine decreased VSMC proliferation and induced macroautophagy/autophagy in an in vivo vascular injury model of restenosis. Furthermore, we studied the effects of caffeine in primary human and mouse aortic VSMCs and immortalized mouse aortic VSMCs. Caffeine decreased cell proliferation, and induced autophagy flux via inhibition of MTOR signaling in these cells. Genetic deletion of the key autophagy gene Atg5, and the Sqstm1/p62 gene encoding a receptor protein, showed that the anti-proliferative effect by caffeine was dependent upon autophagy. Interestingly, caffeine also decreased WNT-signaling and the expression of two WNT target genes, Axin2 and Ccnd1 (cyclin D1). This effect was mediated by autophagic degradation of a key member of the WNT signaling cascade, DVL2, by caffeine to decrease WNT signaling and cell proliferation. SQSTM1/p62, MAP1LC3B-II and DVL2 were also shown to interact with each other, and the overexpression of DVL2 counteracted the inhibition of cell proliferation by caffeine. Taken together, our in vivo and in vitro findings demonstrated that caffeine reduced VSMC proliferation by inhibiting WNT signaling via stimulation of autophagy, thus reducing the vascular restenosis. Our findings suggest that caffeine and other autophagy-inducing drugs may represent novel cardiovascular therapeutic tools to protect against restenosis after angioplasty and/or stent placement.

Laparoscopic management of type VI choledochal cyst with common bile duct stone: report of a case and review of literature.

A young adult male presented with biliary colic and intermittent jaundice for 1 year. Abdomen findings were unremarkable. Routine investigations revealed a raised total bilirubin. On abdominal ultrasonography, common bile duct (CBD) dilatation with multiple stones was noted. On further imaging with magnetic resonance cholangiopancreatography, type I choledochal cyst (CDC) was suspected. A laparoscopic approach was planned. Intraoperatively, dilatation of cystic duct was noted which constitute type VI CDC. Partial malrotation of the gut and accessory right hepatic artery were also noted as incidental finding. Laparoscopic cholecystectomy with CBD exploration and removal of stones, biliary stent placement, cystic duct cyst excision and primary repair of CBD was done. Postoperatively, the patient improved symptomatically with a fall in bilirubin to normal range. We are describing the laparoscopic management of a rare case of type IV CDC which was diagnosed intraoperatively.